Methods of treating chronic inflammatory diseases

ABSTRACT

The invention relates to method of treating chronic inflammatory diseases in subjects in need thereof by administering calcium lactate as an active agent. The calcium lactate can be provided in a pharmaceutical, food, or nutrient composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/246,252 filed Jan. 11, 2019, which claims benefit of the filing dateof U.S. Provisional Application No. 62/616,923, filed Jan. 12, 2018, thecontents of each of which are incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Chronic inflammatory disease(s) (CID) underpins the pathologicalcondition characterized by chronic inflammation, defined as a prolongedand persistent pro-inflammatory state (1). CID includes many common anduncommon CID such as respiratory diseases (e.g., asthma, chronicobstructive pulmonary disease, pneumonitis, and pulmonary fibrosis) (2),ocular diseases (e.g., keratitis and age-related macular degeneration)(3), autoimmune diseases (4), nephropathy (5), neurodegenerativediseases (e.g., Alzheimer's disease, stroke, Parkinson's disease, andmultiple sclerosis) (6), cardiovascular diseases (e.g., atherosclerosis,arteriosclerosis, and myocarditis) (7), metabolic disorders (e.g.,diabetes and obesity) (8), musculoskeletal diseases (e.g., rheumatoidarthritis and osteoporosis) (9, 10), periodontal diseases (e.g.,pulpitis and periodontitis) (11), digestive diseases (e.g.,non-alcoholic fatty liver disease, gastroenteritis, and chronicinflammatory bowel disease) (12), and skin disorders (e.g., psoriasisand atopic dermatitis) (13, 14).

An epidemiological study showed that CID is a leading cause of deaththroughout the world and has been increasing over the last threedecades. It has been estimated that by 2030, 171 million people will beaffected by CID in the United States (15). There are various types ofanti-inflammatory drugs to treat inflammation, such as aspirin,antihistamines, COX-2 inhibitors, corticosteroid, and nonsteroidalanti-inflammatory drugs (NSAIDs). However, there are limitations forlong term administration of such existing drugs due to side effects andtemporal efficacy.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a method of treating a chronic inflammatory disease,the method comprising administering to a subject in need thereof atherapeutically effective amount of calcium lactate.

Disclosed herein is a method of treating Alzheimer's disease,Parkinson's disease, atherosclerosis, and/or stroke, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of calcium lactate.

Also disclosed is a method of treating multiple sclerosis, age-relatedmacular degeneration, nonalcoholic fatty liver disease (NAFLD), and/orrheumatoid arthritis, the method comprising administering to a subjectin need thereof a therapeutically effective amount of calcium lactate.In some embodiments, the method is for treating NAFLD and the NAFLD isnonalcoholic steatohepatitis (NASH).

Also disclosed is a method of treating pulpitis and/or periodontitis,the method comprising administering to a subject in need thereof atherapeutically effective amount of calcium lactate.

Further disclosed is a method of treating inflammatory bowel disease(IBD), the method comprising administering to a subject in need thereofa therapeutically effective amount of calcium lactate. In someembodiments, the IBD is Crohn's disease or ulcerative colitis.

Further disclosed is a method of treating spinal cord injury, cerebralhemorrhage, myocardial infarction, keratitis, and/or diabeticretinopathy, the method comprising administering to a subject in needthereof a therapeutically effective amount of calcium lactate.

Disclosed herein is a method of treating asthma, pulmonary fibrosis,obesity, gastroenteritis, chronic inflammatory bowel disease, and/oratopic dermatitis, the method comprising administering to a subject inneed thereof a therapeutically effective amount of calcium lactate. Alsodisclosed herein is a method of treating chronic obstructive pulmonarydisease, pneumonitis, keratitis, atherosclerosis, arteriosclerosis,myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis,and/or psoriasis, the method comprising administering to a subject inneed thereof a therapeutically effective amount of calcium lactate. Alsodisclosed herein is a method of treating Alzheimer's disease, stroke,Parkinson's disease, multiple sclerosis, age-related maculardegeneration, non-alcoholic fatty liver disease, sepsis, and/orosteoporosis, the method comprising administering to a subject in needthereof a therapeutically effective amount of calcium lactate.

In some embodiments, the calcium lactate is administered in apharmaceutical composition comprising a pharmaceutically acceptablecarrier, excipient, and/or diluent. In some embodiments, thepharmaceutical composition is formulated into liquid, powder, aerosol,injection, fluid transfusion, patch, capsule, pill, tablet, depot, orsuppository.

In some embodiments, the pharmaceutical composition comprises atherapeutically effective amount of calcium lactate as an active agentand a pharmaceutically acceptable polysaccharide, polymer, lipid, orcombinations thereof. In some embodiments, the pharmaceuticalcomposition comprises the calcium lactate and the polysaccharide. Insome embodiments, a weight ratio of the calcium lactate to thepolysaccharide is 1:<0.2 to 1:5. In some embodiments, a weight ratio ofthe calcium lactate to the polysaccharide is 1:<0.2. In someembodiments, a weight ratio of the calcium lactate to the polysaccharideis 1:0.2 to 1:5.

In some embodiments, the pharmaceutical composition further comprises apolymer and/or lipid. In some embodiments, the pharmaceuticalcomposition further comprises the polymer and lipid, wherein the weightratio of the polymer to the lipid is 1:0.1 to 1:50. In some embodiments,the weight ratio of the calcium lactate to the polymer and/or lipid isat least 1:5. In some embodiments, a weight ratio of the calcium lactateto the polymer and/or lipid is 1:5 to 1:30.

In some embodiments, the pharmaceutical composition comprises thecalcium lactate and the polymer and/or lipid. In some embodiments, thepharmaceutical composition comprises the polymer and lipid, wherein theweight ratio of the polymer to the lipid is 1:0.1 to 1:50. In someembodiments, a weight ratio of the calcium lactate to the polymer and/orlipid is at least 1:5. In some embodiments, the weight ratio of thecalcium lactate to the polymer and/or lipid is 1:5 to 1:30.

In some embodiments, the composition is short-acting. In someembodiments, the composition is long-acting. In some embodiments, thecomposition is an injectable composition.

In some embodiments, the polysaccharide is a cellulose derivative,pectin, hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen,alginate, alginic acid or combinations thereof.

In some embodiments, the polymer is a poloxamer, polyvinylpyrrolidone,polyethylene glycol (PEG), polyglycolic lactic acid (PLGA), orcombinations thereof.

In some embodiments, the lipid is a mono- or tri-fatty acid glycerinester or polyethylene glycol, polyethylene glycol esters of vegetableoils, fatty acid propylene glycol esters, sesame oil, soybean oil,castor oil, corn oil, palm oil, peanut oil, cacao oil, cottonseed oil,sunflower seed oil, safflower oil, almond oil, olive oil, hydrogenatedoil, oleic acid, linolenic acid, linoleic acid, palmitic acid,palmitoleic acid, arachadonic acid, myristic acid, capric acid, caprylicacid, lauric acid, stearic acid, ethyl oleate, isopropyl palmitate,octyldodecyl myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol,cetyl alcohol, stearyl alcohol, or combinations thereof.

In some embodiments, upon placement of the pharmaceutical composition inan in vitro dissolution test comprising an elution test method at 300rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylonfilter having a pore size of 45 μm, at least about 40% of the activeagent is released after 6 hours.

In some embodiments, upon placement of the pharmaceutical composition inan in vitro dissolution test comprising an elution test method at 300rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylonfilter having a pore size of 45 μm, at least about 60% of the activeagent is released after 12 hours.

In some embodiments, upon placement of the pharmaceutical composition inan in vitro dissolution test comprising an elution test method at 300rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylonfilter having a pore size of 45 μm, at least about 80% of the activeagent is released after 24 hours.

In some embodiments, upon placement of the pharmaceutical composition inan in vitro dissolution test comprising an elution test method at 300rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylonfilter having a pore size of 45 μm, at least about 90% of the activeagent is released after 48 hours.

In some embodiments, wherein upon placement of the pharmaceuticalcomposition in an in vitro dissolution test comprising an elution testmethod at 300 rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C.using a nylon filter having a pore size of 45 μm, less than about 40% ofthe active agent is released after 24 hours.

In some embodiments, upon placement of the pharmaceutical composition inan in vitro dissolution test comprising an elution test method at 300rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylonfilter having a pore size of 45 μm, less than about 60% of the activeagent is released after 48 hours.

In some embodiments, upon placement of the pharmaceutical composition inan in vitro dissolution test comprising an elution test method at 300rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylonfilter having a pore size of 45 μm, less than about 80% of the activeagent is released after 72 hours.

In some embodiments, upon placement of the pharmaceutical composition inan in vitro dissolution test comprising an elution test method at 300rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylonfilter having a pore size of 45 μm, less than about 90% of the activeagent is released after 144 hours.

In some embodiments, the pharmaceutical composition is contained in asterile glass or polyolefin container.

In some embodiments, the calcium lactate is coated with apharmaceutically acceptable enteric coating. In some embodiments, theenteric coating comprises hydroxypropyl methyl cellulose phthalate(HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate(PVAP), shellac, polymer of methacrylic acid and an ester thereof, orcombinations thereof. In some embodiments, the weight ratio of thecalcium lactate to the enteric coating is 10:0.5 to 1:1.5. In someembodiments, in an in vitro dissolution test comprising a USP Paddlemethod at a paddle speed of 50 rpm at 37° C., when the composition isplaced in 0.1 HCl for 120 minutes followed by adjusting to pH 6.8 withphosphate buffer, less than about 20% of the active agent is releasedafter 30 minutes. In some embodiments, in an in vitro dissolution testcomprising a USP Paddle method at a paddle speed of 50 rpm at 37° C.,when the composition is placed in 0.1 N HCl for 120 minutes followed byadjusting to pH 6.8 with phosphate buffer, less than 30% of the activeagent is released after 60 minutes. In some embodiments, in an in vitrodissolution test comprising a USP Paddle method at a paddle speed of 50rpm at 37° C., when the composition is placed in 0.1 N HCl for 120minutes followed by adjusting to pH 6.8 with phosphate buffer, less than50% of the active agent is released after 120 minutes. In someembodiments, in an in vitro dissolution test comprising a USP Paddlemethod at a paddle speed of 50 rpm at 37° C., when the composition isplaced in 0.1 N HCl for 120 minutes followed by adjusting to pH 6.8 withphosphate buffer, less than 10% of the active agent is released after120 minutes.

In some embodiments, the calcium lactate is provided in a food ornutrient composition comprising calcium lactate. In some embodiments,the food or nutrient composition is an injectable nutritionalsupplement.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Human leukemic monocyte in vitro experiment. Western blotresults indicated that inflammatory factor (NF-κb) was decreased by 2.5mM calcium lactate treatment in THP-1 cells (human leukemic monocyte)under hypoxia condition.

FIG. 2. In vitro experiment of human leukemic monocytes differentiatedinto macrophages. Western blot results indicated that inflammatoryfactor (NF-κb) was decreased by 2.5 mM calcium lactate treatment indifferentiated THP-1 cells with PMA (phorbol-12-myristate-13-acetate)(M0 macrophage) under normoxia and hypoxia conditions.

FIG. 3. Western blot results indicated that inflammatory factor (NF-κb)was decreased by 2.5 mM calcium lactate treatment in differentiatedTHP-1 cells with interferon gamma (IFN-γ) and Lipopolysaccharide (LPS)(M1 macrophage) under normoxia and hypoxia conditions.

FIG. 4. Western blot results indicated that inflammatory factor (NF-κb)was decreased by 2.5 mM calcium lactate treatment in differentiatedTHP-1 cells with interleukin-4 (IL-4) and interleukin-10 (IL-10) (M2macrophage) under normoxia and hypoxia conditions.

FIG. 5. Liver fibrosis in vitro experiment. Western blot resultsindicated that hypoxia inducible factor-1α (HIF-1α) was decreased by 2.5mM calcium lactate treatment in LX-2 cells (human hepatic stellate cell)under hypoxia condition.

FIG. 6. Human endothelia cells in vitro experiment. Western blot resultsindicated that hypoxia-mediated factors (lactate dehydrogenase A) weredecreased by 2.5 mM calcium lactate treatment in human umbilical veinendothelial cells under hypoxia condition.

FIG. 7. Human fibroblast in vitro experiment. Western blot resultsindicated that lactate dehydrogenase B was increased by 2.5 mM calciumlactate treatment in CCD-18Co cells (human colon fibroblast) underhypoxia condition.

FIG. 8. Brain CID in vitro experiment. Western blot results indicatedthat hypoxia-mediated factors were decreased by 2.5 mM calcium lactatetreatment in SK-N-SH cells (brain epithelium) under normoxia and hypoxiaconditions.

FIG. 9. Liver CID in vitro experiment. Western blot results indicatedthat hypoxia-mediated factors were changed by 2.5 mM calcium lactatetreatment in HepG2 cells (liver epithelium) under hypoxia condition.

FIG. 10. Western blot results indicated that inflammatory factor (TLR-4)was decreased by 2.5 mM calcium lactate treatment in HepG2 cells (liverepithelium) under hypoxia condition.

FIG. 11. Eye CID in vitro experiment. Western blot results indicatedthat hypoxia-mediated factors were decreased by 2.5 mM calcium lactatetreatment in ARPE-19 cells (retinal pigment epithelium) under normoxiaand hypoxia conditions.

FIG. 12. Immunocytochemistry results indicated that hypoxia induciblefactor-la (HIF-1α) was decreased by 2.5 mM calcium lactate treatment inARPE-19 cells (retinal pigment epithelium) under hypoxia condition.

FIG. 13. The immunocytochemical results indicated that lactatedehydrogenase A was decreased by 2.5 mM calcium lactate treatment in theARPE-19 cells (retinal pigment epithelium) under normoxia condition.

FIG. 14. The immunocytochemical results indicated that inflammatoryfactor (nuclear factor-kappa B) was decreased by 2.5 mM calcium lactatetreatment in the ARPE-19 cells (retinal pigment epithelium) underhypoxia condition.

FIG. 15. The immunocytochemical results indicated that inflammatoryfactor (toll like receptor 4) was decreased by 2.5 mM calcium lactatetreatment in the ARPE-19 cells (retinal pigment epithelium) underhypoxia condition.

FIG. 16. The immunocytochemical results indicated that lactatedehydrogenase B was increased by 2.5 mM calcium lactate treatment in theARPE-19 cells (retinal pigment epithelium) under hypoxia condition.

FIG. 17. The immunocytochemical results indicated that drusen marker(Apolipoprotein E) was decreased by 2.5 mM calcium lactate treatment inthe ARPE-19 cells (retinal pigment epithelium) under hypoxia condition.

FIG. 18. Western blot results indicated that inflammatory factor (NF-κb)was decreased by 2.5 mM calcium lactate treatment in ARPE-19 cells(retinal pigment epithelium) under hypoxia condition.

FIG. 19. The photos show that there is no toxicity in ARPE-19 cells(retinal pigment epithelium) by 2.5 mM calcium lactate treatment.Calcium lactate only plays an important role in the metabolic change ofinjured epithelial cells.

FIG. 20. Liver CID (NASH) in vivo experiment. The blood chemistryresults indicated that serum aspartate aminotransferase (AST) andalanine aminotransferase (ALT) were significantly decreased by 2 mg/kgcalcium lactate treatment. **P<0.001 vs. methionine-choline deficient(MCD). Results are MEAN±SD.

FIG. 21. Immunohistochemical results show that in the liver steatosiswas prevented by 2 mg/kg calcium lactate treatment.

FIGS. 22A, 22B, and 22C Immunohistochemical results show that in theliver lipid droplets are not present in the 2 mg/kg calciumlactate-treated group, similar to the control group, while they arefound in the methionine-choline deficient (MCD) group.

FIG. 23. Immunohistochemical results show that in the liver immune cellinfiltration is decreased in the 2 mg/kg calcium lactate-treated group,similar to the control group, while immune cell infiltration isincreased in the methionine-choline deficient (MCD) group.

FIG. 24. Immunohistochemical results show that in the liver fibrosis isnot present in the 2 mg/kg calcium lactate-treated group, similar to thecontrol group, while it is present in the methionine-choline deficient(MCD) group.

FIG. 25. Western blot results indicated that inflammatory factor (NF-κB)was decreased by 2.5 mM calcium lactate treatment in the tissue ofretinal pigment epithelium.

FIG. 26. Immunohistochemical results showed that the retinal pigmentepithelium was recovered in a similar morphology to the normal controlepithelium by treatment with 2.5 mM calcium lactate.

FIG. 27. Fluorescence images for lesion of inner layer of choroid onflat-mount by laser-induced age-related macular degeneration (AMD).

FIG. 28. Fluorescence images for lesion of inner layer andneovascularization of choroid on flat-mount by laser-induced age-relatedmacular degeneration (AMD).

FIG. 29. Experimental scheme for establishing Alzheimer's disease andstroke.

FIG. 30. Representative images for brain damage by lipopolysaccharide(LPS). Contralateral: normal region. Ipsilateral: LP S injection.

FIG. 31. Recovery of damaged brain tissue and reduction of microgliainfiltration that recruited upon brain damage.

FIG. 32. Experimental scheme for establishing Parkinson's disease.

FIG. 33. Recovery of dopaminergic neuron by treatment with calciumlactate.

FIG. 34. Experimental scheme for establishing multiple sclerosis.

FIG. 35. Recovery of demyelinated spinal cord and reduction of microgliainfiltration by treatment with calcium lactate.

FIG. 36. Representative histological profiles of aorta tissues. Thetissues in left and middle side are hematoxylin and eosin (H&E)staining. The tissues in right side are oil red o staining.

FIG. 37. Masson's trichrome staining for periodontal tissues.

FIG. 38. Masson's trichrome staining for upper gum.

FIG. 39. Masson's trichrome staining for foot and knee joint tissues.

FIG. 40. Experimental scheme for inducing Crohn's disease.

FIG. 41. Representative images for hematoxylin and eosin (H&E) stainingof Crohn's disease.

FIG. 42. Experimental scheme for inducing colitis.

FIG. 43. Representative images for hematoxylin and eosin (H&E) stainingof colitis.

DETAILED DESCRIPTION OF THE INVENTION

Recently, many studies showed that CID is related to advancing age andsuggested chronic inflammation as being associated with age-relatedimmuno-senescence diseases (1-29). Chronic inflammation was elucidatedto occur via complex interactions between parenchymal cells and stromalcells, such as immune cells, endothelial cells, and fibroblasts. Amongothers, myeloid-originated cells, such as monocytes and activatedmacrophages, were identified as major effector cells in CID. Uponactivation with bacterial lipopolysaccharide, IFN-gamma, and damagedcells, macrophages produced many inflammatory cytokines and clearedendogenous and exogenous inflammatory agents through transcriptionalcontrol by nuclear factor-kappa B (NF-kB). Macrophages were also furtherpolarized to more specialized macrophage subtypes. However, their finelycontrolled differentiation and activation of myeloid cells weredysregulated in chronic inflammation. Local tissue hypoxia caused themetabolic changes of cells, such as parenchymal and stromal cells,through induction of hypoxia inducible factors (HIFs). HIFs, especiallyHIF-1, contribute to the abnormal activation of macrophages and are amajor contributor to chronic inflammatory response.

Most CID are characterized by inflammation. Injured tissue becomeresistant to blood flow and delivery of oxygen, thus becoming a hypoxicenvironment. Proinflammatory mediators and a hypoxic condition canelicit an angiogenic response through the induction of HIF-1.Additionally, a local inflammatory reaction exists in most CID,including as respiratory diseases (e.g., asthma, chronic obstructivepulmonary disease, pneumonitis, and pulmonary fibrosis), ocular diseases(e.g., keratitis and age-related macular degeneration), autoimmunediseases, nephropathy, neurodegenerative diseases (e.g., Alzheimer'sdisease, stroke, Parkinson's disease, and multiple sclerosis),cardiovascular diseases (e.g., atherosclerosis, arteriosclerosis, andmyocarditis), metabolic disorders (e.g., diabetes and obesity),musculoskeletal diseases (e.g., rheumatoid arthritis and osteoporosis),periodontal diseases (e.g., pulpitis and periodontitis), digestivediseases (e.g., non-alcoholic fatty liver disease, gastroenteritis, andchronic inflammatory bowel disease), and skin disorders (e.g., psoriasisand atopic dermatitis). During inflammation, vascular permeability isincreased and monocytes, macrophages, platelets, mast cells, and otherleukocytes are recruited under the attraction of chemokines. Therefore,disclosed herein are therapeutic approaches targeting activation ofmacrophages and regulation of extracellular matrix degradation bytargeting HIF-1, NF-kB, and lactate dehydrogenase (LAD) A and B.

Methods of Treatment

Disclosed herein is a method of treating a chronic inflammatory disease,the method comprising administering to a subject in need thereof atherapeutically effective amount of calcium lactate.

Disclosed herein is a method of treating Alzheimer's disease,Parkinson's disease, atherosclerosis, and/or stroke, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of calcium lactate.

Also disclosed is a method of treating multiple sclerosis, age-relatedmacular degeneration, nonalcoholic fatty liver disease (NAFLD), and/orrheumatoid arthritis, the method comprising administering to a subjectin need thereof a therapeutically effective amount of calcium lactate.In some embodiments, the method is for treating NAFLD and the NAFLD isnonalcoholic steatohepatitis (NASH).

Also disclosed is a method of treating pulpitis and/or periodontitis,the method comprising administering to a subject in need thereof atherapeutically effective amount of calcium lactate.

Further disclosed is a method of treating inflammatory bowel disease(IBD), the method comprising administering to a subject in need thereofa therapeutically effective amount of calcium lactate. In someembodiments, the IBD is Crohn's disease or ulcerative colitis.

Further disclosed is a method of treating spinal cord injury, cerebralhemorrhage, myocardial infarction, keratitis, and/or diabeticretinopathy, the method comprising administering to a subject in needthereof a therapeutically effective amount of calcium lactate.

Disclosed herein are methods of treating asthma, pulmonary fibrosis,obesity, gastroenteritis, chronic inflammatory bowel disease, and/oratopic dermatitis, the method comprising administering to a subject inneed thereof a therapeutically effective amount of calcium lactate. Alsodisclosed herein are methods of treating chronic obstructive pulmonarydisease, pneumonitis, keratitis, atherosclerosis, arteriosclerosis,myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis,and/or psoriasis, the method comprising administering to a subject inneed thereof a therapeutically effective amount of calcium lactate. Alsodisclosed herein are methods of treating Alzheimer's disease, stroke,Parkinson's disease, multiple sclerosis, age-related maculardegeneration, non-alcoholic fatty liver disease, and/or sepsis, themethod comprising administering to a subject in need thereof atherapeutically effective amount of calcium lactate.

Also disclosed herein are methods of treating osteoporosis, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of calcium lactate.

The term “subject” or “patient” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of themammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. In some embodiments of the methods andcompositions provided herein, the mammal is a human or a nonhuman.

As used herein, “treat”, “treating”, or “treatment” of one or more ofthe diseases disclosed herein by administration of the pharmaceuticalcomposition refers to any lessening of severity, delay in onset, slowingof progression, or shortening of duration of one or more of the diseasesdisclosed herein, whether permanent or temporary, lasting or transientthat can be attributed to or associated with administration of thecomposition.

The term “co-administration” or the like, as used herein, is meant toencompass administration of the selected two or more active agents to asingle patient and is intended to include treatment regimens in whichthe agents are administered by the same or different route ofadministration or at the same or different time.

The pharmaceutical composition of the present disclosure can beadministered in a therapeutically effective amount, and as used herein,the term “effective amount” or therapeutically effective amount” refersto an amount sufficient to treat or prevent diseases, at a reasonablebenefit/risk ratio applicable to any medical treatment or prevention.The effective dosage level can be determined depending on severity ofthe disease, activity of the drug or active agent, a patient's age, bodyweight, health and sex, sensitivity to the drug, administration time,administration route, and excretion rate of the composition of thepresent disclosure, duration of treatment, drugs used simultaneously orin combination with the composition of the present disclosure, and otherfactors known in the medical field. The pharmaceutical composition ofthe present disclosure can be administered alone or in combination withother publicly-known drugs or components known as known for treating oneor more of the diseases disclosed herein. It is important to administerthe composition in the minimum amount that can exhibit the maximumeffect without causing side effects, in consideration of all the abovefactors.

The pharmaceutical composition of the present disclosure can beadministered such that the dosage per day of calcium lactate is, forexample, about 10 mg/kg to about 1,000 mg/kg, about 10 mg/kg to about500 mg/kg, about 10 mg/kg to about 250 mg/kg, about 10 mg/kg to about200 mg/kg, about 10 mg/kg to about 100 mg/kg, about 1 mg/kg to about 100mg/kg, about 1 mg/kg to about 75 mg/kg, about 1 mg/kg to about 50 mg/kg,about 1 mg/kg to about 25 mg/kg, or about 1 mg/kg to about 10 mg/kg. Theadministration frequency of the composition can be, but is notparticularly limited to, once, twice, three times, four times, etc.divided doses a day.

The compositions containing the compound(s) described herein can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the compositions are administered to a patientalready suffering from a disease or condition, in an amount sufficientto cure or at least partially arrest the symptoms of the disease orcondition. Amounts effective for this use will depend on the severityand course of the disease or condition, previous therapy, the patient'shealth status, weight, and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (including, but not limited to, a doseescalation clinical trial).

In prophylactic applications, compositions containing the active agentdescribed herein are administered to a patient susceptible to orotherwise at risk of one or more of the diseases disclosed herein. Suchan amount is defined to be a “prophylactically effective amount ordose.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial). When used in a patient, effective amounts for this use willdepend on the severity and course of the disease, disorder or condition,previous therapy, the patient's health status and response to the drugs,and the judgment of the treating physician.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the active agent describedherein can be administered chronically, that is, for an extended periodof time, including throughout the duration of the patient's life inorder to ameliorate or otherwise control or limit the symptoms of thepatient's disease or condition.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, diseasecondition and its severity, the identity (e.g., age, weight, gender,etc.) of the subject or host in need of treatment, but can neverthelessbe routinely determined in a manner known in the art according to theparticular circumstances surrounding the case, including, e.g., thespecific agent being administered, the route of administration, thecondition being treated, and the subject or host being treated.

Pharmaceutical Compositions

The term “calcium lactate” refers to a type of lactate metal salt thatcan, for example, exist as a hydrate, represented by C₆H₁₀O₆Ca.5H₂O inwhich calcium ion is bonded to lactate. Calcium lactate can be in theform of white powder or granules at room temperature, or anhydrous at a120° C. heating condition, and has a solubility of 5% (w/v).

Calcium lactate can be formulated into pharmaceutical compositions fortreating one or more of the diseases disclosed herein.

In various embodiments, the invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of calciumlactate as an active agent for treating one or more of the diseasesdisclosed herein and a pharmaceutically acceptable polysaccharide,polymer, lipid, or combinations thereof. In some embodiments, thepharmaceutical composition comprises the calcium lactate and thepolysaccharide.

In some embodiments, the invention provides an enteric coating of thecalcium lactate such that the active agent is protected from the acidicenvironment of the stomach and absorbed in the small intestine before itreaches the large intestine when the active agent is administeredorally.

The present invention also provides short-acting and long-actingpharmaceutical compositions comprising calcium lactate. In someembodiments, the long-acting compositions comprise calcium lactatedcoated with at least one enteric-coating material such as hydroxypropylmethyl cellulose phthalate (HPMCP), cellulose acetate phthalate (CAP),polyvinyl acetate phthalate (PVAP), shellac and a polymer of methacrylicacid, and an ester thereof.

The pharmaceutical composition of the invention can be formulated intopharmaceutical preparations for oral administration. Examples of thepreparation include powders, tablets, capsules, granules or syrups,tablets and capsules, but not limited thereto.

Also provided herein are formulations of hydrogels particularlymethylcellulose, poloxamer, pectin, and alginate hydrogel which canremain in a solution or nanoparticle form in vitro, and the gel can formwhen injected into the body and allow sustained release of calciumlactate. The relatively short drug release time which is the weakness ofthe hydrogel has been improved by increasing the interaction between thedrug and hydrogel or by delaying the diffusion of the drug in thehydrogel.

The weight ratio of the calcium lactate to the polysaccharide can be,e.g., 1:<0.2 to 1:5, 1:0.01 to 1:5, 1:0.05 to 1:5, or 1:0.1 to 1:5. Theweight ratio of the calcium lactate to the polysaccharide can be 1:<0.2.The weight ratio of the calcium lactate to the polysaccharide can be1:0.2 to 1:5.

In some embodiments, the pharmaceutical composition further comprises apolymer or lipid. The weight ratio of the calcium lactate to the polymeror lipid can be at least 1:5. The weight ratio of the calcium lactate tothe polymer or lipid can be 1:5 to 1:30, e.g., 1:5 to 1:30, 1:5 to 1:20,1:5 to 1:10, 1:10 to 1:30, 1:10 to 1:20, or 1:20 to 1:30.

In some embodiments, the pharmaceutical composition further comprises apolymer and lipid. The weight ratio of the calcium lactate to thepolymer and lipid can be at least 1:5. The weight ratio of the calciumlactate to the polymer and lipid can be 1:5 to 1:30, e.g., 1:5 to 1:30,1:5 to 1:20, 1:5 to 1:10, 1:10 to 1:30, 1:10 to 1:20, or 1:20 to 1:30.

In some embodiments, the pharmaceutical composition comprises thecalcium lactate and the polymer or lipid. The weight ratio of thecalcium lactate to the polymer or lipid can be at least 1:5. The weightratio of the calcium lactate to the polymer or lipid can be 1:5 to 1:30.The weight ratio of the calcium lactate to the polymer or lipid can be1:5 to 1:30, e.g., 1:5 to 1:30, 1:5 to 1:20, 1:5 to 1:10, 1:10 to 1:30,1:10 to 1:20, or 1:20 to 1:30.

In some embodiments, the pharmaceutical composition comprises thecalcium lactate and the polymer and lipid. The weight ratio of thecalcium lactate to the polymer and lipid can be at least 1:5. The weightratio of the calcium lactate to the polymer and lipid can be 1:5 to1:30. The weight ratio of the calcium lactate to the polymer and lipidcan be 1:5 to 1:30, e.g., 1:5 to 1:30, 1:5 to 1:20, 1:5 to 1:10, 1:10 to1:30, 1:10 to 1:20, or 1:20 to 1:30.

In some embodiments, the weight ratio of the polymer to the lipid can be1:0.1 to 1:50, 1:0.1 to 1:20, 1:0.1 to 1:10, 1:0.1 to 1:5, 1:0.1 to 1:2,1:0.1 to 1:1, 1:0.1 to 0.5, or 1:0.1 to 1:0.2.

Polysaccharides suitable for use in the composition can be a cellulosederivative (e.g., carboxymethyl cellulose (CMC), ethyl cellulose (EC),hydroxypropyl methyl cellulose (HPMC), methyl cellulose (MC)), pectin,hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen,alginate, alginic acid, or combinations thereof.

Polymers suitable for use in the composition can be a poloxamer,polyvinylpyrrolidone, polyethylene glycol (PEG), polyglycolic lacticacid (PLGA), or combinations thereof.

Lipids suitable for use in the composition can be a mono- or tri-fattyacid glycerin ester or polyethylene glycol, polyethylene glycol estersof vegetable oils, fatty acid propylene glycol esters, sesame oil,soybean oil, castor oil, corn oil, palm oil, peanut oil, cacao oil,cottonseed oil, sunflower seed oil, safflower oil, almond oil, oliveoil, hydrogenated oil, oleic acid, linolenic acid, linoleic acid,palmitic acid, palmitoleic acid, arachadonic acid, myristic acid, capricacid, caprylic acid, lauric acid, stearic acid, ethyl oleate, isopropylpalmitate, octyldodecyl myristate, acetyl palmitate, lauryl alcohol,oleyl alcohol, acetyl alcohol, stearyl alcohol, or combinations thereof.

The invention is further directed to a pharmaceutical compositioncomprising a therapeutically effective amount of calcium lactate as anactive agent for treating one or more of the diseases disclosed herein,wherein the calcium lactate is coated with a pharmaceutically acceptableenteric coating. In some embodiments, the enteric coating compriseshydroxypropyl methyl cellulose phthalate (HPMCP), cellulose acetatephthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymer ofmethacrylic acid and an ester thereof, or combinations thereof. In someembodiments, the weight ratio of the calcium lactate to the entericcoating is 10:0.5 to 10:15, 10:0.5 to 1:1, 10:0.5 to 10:5, 10:0.5 to10:3, 10:0.5 to 10:2, 10:0.5 to 10:1, 10:0.5 to 1:0.8.

By “pharmaceutically acceptable,” as used herein, refers a material,such as a carrier or diluent, which does not abrogate the biologicalactivity or properties of the compound, and is relatively nontoxic,i.e., the material can be administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

A pharmaceutical composition, as used herein, refers to a mixture ofcalcium lactate with other chemical components that are pharmaceuticallyacceptable, such as but not limited to carriers, stabilizers, diluents,disintegrants, suspending agents, thickening agents, binders,antimicrobial agents, antimicrobial preservatives, antioxidants, and/orbuffering agents. The pharmaceutical composition facilitatesadministration of the calcium lactate to a subject.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues. The term “diluent” refers to chemicalcompounds that are used to dilute the compound of interest prior todelivery. Diluents can also be used to stabilize compounds because theycan provide a more stable environment. Pharmaceutically acceptableadditives include diluents, binders, solubilizers, solubility enhancers,pore formers, osmotic agents, gas formers, lubricants and fluidizerswell known in the art, but not limited thereto.

Diluents can include lactose, fructose, dextrose, sucrose, maltose,microcrystalline cellulose, starch, calcium hydrogen phosphate, mannitolor a mixture thereof, but not limited thereto. Other diluents includemicrocrystalline cellulose, lactose, mannitol, calcium phosphate and thelike.

Examples of binders can include povidone, hydroxypropylcellulose,polyvinylalcohol, hydroxypropylmethylcellulose, carboxymethyl-cellulosesodium and thereof.

Solubilizing agents include surfactants, cyclodextrins and derivativesthereof, lipophilic substances or mixtures thereof, but are not limitedthereto.

Surfactants include water soluble or water dispersible nonionic,nonpolar nonionic, anionic, cationic, amphoteric or ionic surfaceactivators or mixtures thereof, but are not limited thereto.

Examples of disintergrants include crospovidone, croscarmellose sodium,glycolic acid starch sodium, and examples of the lubricant includemagnesium stearate, calcium stearate, sodium stearyl fumarate andthereof.

The pharmaceutical compositions of the invention can further includeantimicrobial agents, such as benzyl alcohol, chlorobutanol, phenylethylalcohol, phenyl-mercuric acetate, potassium sorbate, and sorbic acid.Antifungal agents include such compounds as benzoic acid, butylparaben,ethylparaben, methylparaben, propylparaben, and sodium benzoate.

Antimicrobial preservatives can be added to the pharmaceuticalcompositions of the present invention in order to protect them againstthe growth of potentially harmful microorganisms, which usually invadethe aqueous phase, but in some cases can also grow in the oil phase of acomposition. Thus, preservatives with both aqueous and lipid solubilityare desirable. Suitable antimicrobial preservatives include, e.g., alkylesters of p-hydroxybenzoic acid, propionate salts, phenoxyethanol,methylparaben sodium, propylparaben sodium, sodium dehydroacetate,benzalkonium chloride, benzethonium chloride, benzyl alcohol, hydantoinderivatives, quaternary ammonium compounds and cationic polymers,imidazolidinyl urea, diazolidinyl urea, and trisodium ethylenediaminetetracetate (EDTA).

Antioxidants can be added to protect all of the ingredients of thepharmaceutical compositions from damage or degradation by oxidizingagents present in the composition itself or the use environment, e.g.,anoxomer, ascorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, potassium metabisulfite,propyloctyl and dodecyl gallate, sodium metabisulfite, sulfur dioxide,and tocopherols.

Buffering agents can be used to maintain a desired pH of thepharmaceutical compositions once established, from the effects ofoutside agents and shifting equilibria of components of the composition.

The pharmaceutical compositions described herein can be preparedfollowing techniques known in the art, for example, in Remington: TheScience and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated byreference in their entirety.

In some embodiments, the pharmaceutical compositions of the inventionare short-acting. The term “short-acting” refers to a composition thatreleases substantially all of the active agent within 48 hours whentested in an in vitro dissolution test described herein, for example,from time of delivery, time 0, until about 1 hour to about 48 hours,until about 3 hours to about 24 hours, until about 6 hours to about 24hours, or until about 12 hours to about 24 hours.

For example, upon placement of the composition in an in vitrodissolution test comprising an elution test method (e.g., from LabfineCo., Mumbai, India) at 300 rpm in 200 ml aqueous medium having a pH of6.8 at 37° C. using a nylon filter having a pore size of 45 μm, at leastabout 40% of the active agent is released after 6 hours, at least about60% of the active agent is released after 12 hours, at least about 80%of the active agent is released after 24 hours, and/or at least about90% of the active agent is released after 48 hours. In some embodiments,upon placement of the composition in an in vitro dissolution testcomprising an elution test method (e.g., from Labfine Co., Mumbai,India) at 300 rpm in 200 ml aqueous medium having a pH of 6.8 at 37° C.using a nylon filter having a pore size of 45 μm, at least about 40% toabout 60% of the active agent is released after 6 hours, at least about60% to about 80% of the active agent is released after 12 hours, atleast about 80% to about 90% of the active agent is released after 24hours, and/or at least about 90% to about 100% of the active agent isreleased after 48 hours.

In some embodiments, the pharmaceutical compositions of the inventionare long-acting. The term “long-acting” is intended to mean compositionthat releases the active agent slowly after the initial dosage, forexample, from time of delivery, time 0, until about 48 hours to about192 hours, until about 72 hours to about 192 hours, until about 96 hoursto about 192 hours, until about 120 hours to about 192 hours, or untilabout 144 hours to about 192 hours.

In some embodiments, upon placement of the composition in an in vitrodissolution test comprising an elution test method (e.g., from LabfineCo., Mumbai, India) at 300 rpm in 200 ml aqueous medium having a pH of6.8 at 37° C. using a nylon filter having a pore size of 45 μm, lessthan about 40% of the active agent is released after 24 hours, less thanabout 60% of the active agent is released after 48 hours, less thanabout 80% of the active agent is released after 72 hours, less thanabout 90% of the active agent is released after 144 hours. In someembodiments, upon placement of the composition in an in vitrodissolution test comprising an elution test method (e.g., from LabfineCo., Mumbai, India) at 300 rpm in 200 ml medium having a pH of 6.8 at37° C. using a nylon filter having a pore size of 45 μm, about 20% toabout 50% of the active agent is released after 24 hours, about 20% toabout 40% of the active agent is released after 24 hours, about 40% toabout 70% of the active agent is released after 48 hours, about 40% toabout 60% of the active agent is released after 48 hours, about 40% toabout 80% of the active agent is released after 72 hours, about 50% toabout 80% of the active agent is released after 72 hours, about 60% toabout 90% of the active agent is released after 144 hours, or about 70%to about 90% of the active agent is released after 144 hours.

In some embodiments, upon placement of the composition in an in vitrodissolution test comprising a USP Paddle method at a paddle speed of 50rpm at 37° C., when the composition is placed in 0.1 N HCl for 120minutes followed by adjusting to pH 6.8 with phosphate buffer, less thanabout 20% of the active agent is released after 30 minutes, less than30% of the active agent is released after 60 minutes, less than 50% ofthe active agent is released after 120 minutes, and/or less than 10% ofthe active agent is released after 120 minutes.

In some embodiments, the pharmaceutical compositions of the inventionare injectable dosage forms. For parenteral injections, appropriateformulations can include aqueous or nonaqueous solutions, preferablywith physiologically compatible carriers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compound(s) in water-soluble form.Additionally, suspensions of the active agent can be prepared asappropriate oily injection suspensions. Suitable lipids or lipophiliccarriers include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. The lipidcan be a mono- or tri-fatty acid glycerin ester or polyethylene glycol,polyethylene glycol esters of vegetable oils, fatty acid propyleneglycol esters, sesame oil, soybean oil, castor oil, corn oil, palm oil,peanut oil, cacao oil, cottonseed oil, sunflower seed oil, saffloweroil, almond oil, olive oil, hydrogenated oil, oleic acid, linolenicacid, linoleic acid, palmitic acid, palmitoleic acid, arachadonic acid,myristic acid, capric acid, caprylic acid, lauric acid, stearic acid,ethyl oleate, isopropyl palmitate, octyldodecyl myristate, cetylpalmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearylalcohol, or combinations thereof.

Since the medical application of hydrogels, numerous hydrogels have beendeveloped and studied in many fields, including the medical,pharmaceutical, and cosmetic industries. Although hydrogels aregenerally biocompatible, they have various problems that are limitingthe delivery of a drug, and various efforts are made to solve theproblems. A hydrogel is a three-dimensional structure composed of anetwork of hydrophilic polymers. More than 90% of the components arecomposed of water. Hydrogels have been actively studied in thebiomedical field due to their similarity to bio-tissue such as highmoisture content, porous structure, relatively soft properties, andbiocompatibility. Hydrogels can exhibit various properties depending onthe kind of polymer used as the main chain or the crosslinking methodadopted. When a polymer of polyacrylic acid series polymer or asynthetic compound such as polyvinyl alcohol is used, thebiocompatibility is low, but the chemical modification is easy, so thatthe engineering application is very easy. On the other hand, whennatural compounds, especially pectin, alginate, collagen, fibrin andhyaluronic are used as the main chain, the chemical modification isdifficult. Nevertheless, there are advantages of using these materialsthat are biologically derived components, as it is suitable for clinicalapplication and there are few side effects such as immune inflammationreaction at the time of transplantation.

It is the cross-linking method that affects the properties of thehydrogel as well as the type of polymer used. Even if the same polymeris used as the main chain, the hydrogel having completely differentcharacteristics can be obtained if the crosslinking method is different.The method of crosslinking hydrogels can be broadly divided intophysical and chemical methods. Physical crosslinking methods includeionic interaction, hydrophobic interaction, hydrogen bond, andreversible crosslinking by structural entanglement. These crosslinkingmethods can easily induce the formation of a three-dimensional networkstructure without the need for a separate chemical additive orcomplicated process. On the other hand, the chemical crosslinking methodtypically forms covalent bond that result an irreversible and stablenetwork as compared with the physical crosslinking method. Hydrogelswith excellent biocompatibility and various physicochemical propertieshave been extensively studied in biomedical fields such as drug deliveryand tissue engineering. Most hydrogels exhibit shorter drug release timethan other drug delivery systems due to their high water content, andthere is a need to develop a system with a longer drug release time.

In some embodiments, the pharmaceutical compositions of the inventionare oral dosage forms. For oral administration, the compound describedherein can be formulated readily by combining the active agent withpharmaceutically acceptable carriers or excipients well known in theart. Such carriers enable the active agent described herein to beformulated as tablets, powders, pills, dragees, capsules, liquids, gels,syrups, elixirs, slurries, suspensions and the like, for oral ingestionby a patient to be treated.

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid carriers with the compound described herein, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients include, but are not limited to,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as: for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents can be added, such as the cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions can be formulated in conventional mannerusing one or more pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activeagent into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques can be used as suitable and as understood inthe art. Pharmaceutical compositions described herein can bemanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

Aqueous suspensions can also contain one or more polymers as suspendingagents. The polymer can be a poloxamer, polyvinylpyrrolidone,polyethylene glycol (PEG), polyglycolic lactic acid (PLGA), orcombinations thereof. Other useful polymers include water-solublepolymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. Useful compositions can also include amucoadhesive polymer, selected from, for example,carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate, and dextran.

In some embodiments, provided are pharmaceutical dosage forms comprisingcalcium lactate and a pharmaceutically acceptable enteric coating inorder to control the release of the active agent. In some embodiments,the coating is a film and, in another embodiment, it is a membrane. Theenteric coating, e.g., film or membrane, can serve to delay releaseuntil after the stomach and to protect the active agent from gastricfluid. The enteric coating can comprise one or more substancespreferably of a polymeric nature (e.g., methacrylates etc.;polysaccharides etc. as described in more detail below) or combinationof more than one such substance, optionally including other excipients,such as, for example, plasticizers. In some embodiments, the entericcoating comprises hydroxypropyl methyl cellulose phthalate (HPMCP),cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP),shellac, polymer of methacrylic acid and an ester thereof, orcombinations thereof. In some embodiments of the invention thecomposition comprises a hydrogel-forming polymer and further polymersable to achieve a desired delay (or other change) in the release of theactive agent.

In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch.

The sterile injectable preparation can also be a sterile injectablesuspension in a non-toxic parenterally acceptable carrier such aslipids.

The amount of calcium lactate included in the pharmaceuticalcompositions can be, but is not limited to, from 1 wt % to 50 wt %, from1 wt % to 40 wt %, from 1 wt % to 35 wt %, from 1 wt % to 30 wt %, from1 wt % to 20 wt %, from 1 wt % to 15 wt %, or from 1 wt % to 10 wt %based on the total weight of the final composition. The concentration ofthe calcium lactate included in a single dose of the pharmaceuticalcomposition can be, but is not limited to, 2.5 mM to 100 mM, 2.5 mM to50 mM, 2.5 mM to 25 mM, 5 mM to 100 mM, 5 mM to 50 mM, 5 mM to 25 mM, 10mM to 100 mM, 10 mM to 50 mM, or 10 mM to 25 mM.

Solid oral pharmaceutical compositions can be prepared by conventionaltechniques such as dry granulation, direct compression, wet granulation,extrusion spheronization, melt granulation or compression coating, butnot limited thereto. The coating can be applied as described below andcan vary as to thickness and density. The amount of coating is definedby the additional weight added to (gained by) the dry composition (e.g.,bead or core containing the calcium lactate) of the invention. Weightgain can be in the range of 0.1% to 50%, 1% to 20%, 1% to 15%, 3% to10%, 5% to 12%, or 8% to 12%.

The coating process can be carried out by any suitable means such as,for example, by use of a coating machine which applies a solution of apolymer coat (as described above in particular) to the composition.Polymers for coating are either provided by the manufacturer inready-made solutions for direct use or can be made up before usefollowing manufacturers' instructions.

Unit Dosages and Kits

The term “unit dosage form” refers to a physically discrete unitsuitable as a single dosage, each unit containing a predeterminedquantity of active ingredient, in association with a suitablepharmaceutical excipient, by which one or more is used throughout thedosing regimen to produce a desired therapeutic effect, e.g., treatingone or more of the diseases disclosed herein.

The pharmaceutical compositions described herein can be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more active agents. The unit dosage canbe in the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection can be presented in unitdosage form, which include, but are not limited to ampoules, or inmulti-dose containers.

The daily dosages appropriate for calcium lactate can be from about 1mg/kg to about 1000 mg/kg, about 10 mg/kg to about 750 mg/kg, about 10mg/kg to about 500 mg/kg, or about 100 mg/kg to 500 mg/kg per bodyweight. An indicated daily dosage in the larger mammal, including, butnot limited to, humans, is in the range from about 5 mg to about 100,000mg, conveniently administered in divided doses, including, but notlimited to, up to four times a day or in long-acting form. Suitable unitdosage forms for administration include from about 10 mg to about 1000mg, about 100 mg to about 1000 mg, about 500 mg to about 750 mg, about25 mg to about 250 mg, about 50 mg to about 100 mg, about 10 mg to about200 mg, or about 10 mg to about 250 mg of the active agent. Theadministration frequency of the composition of the present disclosurecan be, but is not particularly limited to, once, twice, three times,four times, etc. divided doses a day. An appropriate “effective” amountin any individual case can be determined using techniques, such as adose escalation study.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagescan be altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD₅₀ and ED₅₀. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of the active agent liespreferably within a range of circulating concentrations that include theED₅₀ with minimal toxicity. The dosage can vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

According to the present invention, the pharmaceutical compositions canbe in the form of a sterile injectable preparation, for example asterile injectable aqueous or oleaginous suspension. The pharmaceuticalcompositions of the present invention can be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, aqueous suspensions or solutions.

The invention is also directed to a sterile glass or polyolefincontainer comprising a pharmaceutical composition disclosed herein. Insome embodiments, the container is non-DEHP (Bis(2-ethylhexyl) phthalate(di-2-ethylhexyl phthalate, diethylhexyl phthalate, DEHP; dioctylphthalate, DOP) or non-PVP (Polyvinylpyrrolidone).

A kit comprises suitable containers, such as boxes, individual bottles,bags or ampoules. Suspension compositions can be packaged in single-dosenon-reclosable containers or multiple-dose reclosable containers.

Incorporated herein by reference in their entirety are US2017/0360727A1,published Dec. 21, 2017, and PCT/M2017/054091, Int'l Filing Date: Jul.7, 2017. Also incorporated herein by reference in their entirety areU.S. Appl. No. 62/616,923 filed Jan. 12, 2018.

EXAMPLES Example 1. Cell Culture for Age-Related Macular Disease

Human retinal pigment epithelium cell line, ARPE-19, was purchased fromAmerican Type Culture Collection (Manassas, Va., USA). The ARPE-19 cellswere maintained in RPMI1640 containing 10% fetal bovine serum (LifeTechnologies, Gaithersburg, Md., USA), 100 IU/ml penicillin, and 100μg/ml streptomycin (Welgene, Daegu, South Korea).

Example 2. Cell Culture for Alzheimer's Disease

Human Brain cell line, SK-N-SH, was purchased from American Type CultureCollection (Manassas, Va., USA). The SK-N-SH cells were maintained inRPMI1640 containing 10% fetal bovine serum (Life Technologies,Gaithersburg, Md., USA), 100 IU/ml penicillin, and 100 μg/mlstreptomycin (Welgene, Daegu, South Korea).

Example 3. Cell Culture for Fibroblast

Human Colon Fibroblast cell line, CCD-18-Co, was purchased from AmericanType Culture Collection (Manassas, Va., USA). The CCD-18Co cells weremaintained in DMEM containing 10% fetal bovine serum (Life Technologies,Gaithersburg, Md., USA), 100 IU/ml penicillin, and 100 μg/mlstreptomycin (Welgene, Daegu, South Korea).

Example 4. Cell Culture for Non-Alcoholic Steatohepatitis (NASH)

Human Hepatocellular carcinoma cell line, HepG2, was purchased fromAmerican Type Culture Collection (Manassas, Va., USA). The HepG2 cellswere maintained in RPMI 1640 containing 10% fetal bovine serum (LifeTechnologies, Gaithersburg, Md., USA), 100 IU/ml penicillin, and 100μg/ml streptomycin (Welgene, Daegu, South Korea).

Example 5. Cell Culture for Vascular Disease

Human Umbilical Vein Endothelial cell line, HUVEC, was purchased fromAmerican Type Culture Collection (Manassas, Va., USA). The HUVEC cellswere Endothelial Cell Growth Medium 2 maintained in Endothelial CellGrowth Medium 2 Supplement MIX (PromoCell, Heidelberg, Germany)

Example 6. Macrophage Culture

Human Hepatic Stellate cell line, LX-2, was purchased from Sigma-Aldrich(St Louis, Mo., USA). The LX-2 cells were maintained in DMEM containing2% fetal bovine serum (Life Technologies, Gaithersburg, Md., USA), 100IU/ml penicillin, and 100 μg/ml streptomycin (Welgene, Daegu, SouthKorea).

Human Monocyte cell line, THP-1, was purchased from American TypeCulture Collection (Manassas, Va., USA). The THP-1 cells were maintainedin RPMI1640 containing 10% fetal bovine serum (Life Technologies,Gaithersburg, Md., USA), 100 IU/ml penicillin, 100 μg/ml streptomycin(Welgene, Daegu, South Korea), and 0.55 uM 2-Mecaptpethanol. Cell werecultured in both conditions, one condition is a humidified atmosphere at37° C. containing 5% CO₂, and another is hypoxia culture condition wasmaintained at 1% oxygen, 5% and 94%.

Example 7. Regents

Calcium lactate (CaLa) and lipopolysaccharide (LPS) were purchased fromSigma-Aldrich (St Louis, Mo., USA). 2-mercaptoethanol was purchased fromGibco (Grand Island, N.Y., USA). Phorbol 12-myristate 13-acetate (PMA)was purchased from Enzo Lifesciences (Enzo Diagnostic, NY, USA). HumanInterferon-gamma, Human Interleukin-4 (IL-4) and Human Interleukin-10(IL-10) were purchased from BPS Bioscience (San Diego, Calif., USA).Paraformaldehyde (PFA) solution (4%) was purchased from Biosesang(Biosesang Inc., Gyeonggi, Korea).

Example 8. Cell Viability Assay

The live cell movie analyzer, JuLI™ Br (NanoEnTek Inc., Seoul, Korea) inARPE-19 cells, was used to confirm the toxicity of CaLa. Cells wereseeded into 6-well culture plates at a density of 2.5×10⁵ cells/well inmedium. After 24 h incubation, the cells treated with 2.5 mM CaLa for 12hr and 24 hr. The cells were continuously monitored for 24 h during CaLatreatment time. (30, 31)

Example 9. Macrophage Differentiation

The human monocytic cell line, THP-1, was purchased from American TypeCulture Collection (Manassas, Va., USA). THP-1 cells were maintained inRPMI 1640 media (Hyclone Laboratories, Inc., Logan, Utah, USA)supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc.,Logan, Utah, USA), 100 IU/ml penicillin (Welgene, Daegu, South Korea),100 μg/ml streptomycin (Welgene, Daegu, South Korea), and 0.55 μM2-mercaptoethanol (Gibco, Grand Island, N.Y., USA). THP-1 cell wasmaintained in a humidified atmosphere of 5% CO2, at 37° C. THP-1monocytes are differentiated into macrophages by 12 h incubation withPhorbol 12-myristate 13-acetate (PMA) and 100 μg/ml (Enzo Diagnostic,NY, USA) in RPMI 1640 medium. Macrophages were polarized in M1macrophages by 24 h incubation with 20 ng/ml of IFN-γ (BPS Bioscience,San Diego, Calif., USA) and 100 μg/ml of LPS (Sigma-Aldrich, St Louis,Mo., USA) in RPMI 1640 medium. Macrophage M2 polarization was obtainedby 24 h incubation with 20 ng/ml of IL-4 (BPS Bioscience, San Diego,Calif., USA) and 20 ng/ml of IL-10 (BPS Bioscience, San Diego, Calif.,USA) in RPMI 1640 medium. (32)

Example 10. Optical Observation for Cell Viability

The live cell movie analyzer, JuLI™ Br (NanoEnTek Inc., Seoul, Korea) inARPE-19 cells, was used to confirm the toxicity of CaLa. Cells wereseeded into 6-well culture plates at a density of 2.5×10⁵ cells/well inmedium. After a 24 h incubation, the cells treated with 2.5 mM CaLa for12 h and 24 hr. The cells were continuously monitored for 24 h duringCaLa treatment time.

Example 11. Western Blot Analysis

To prepare whole cell lysate, cells were lysed with lysis buffer (1%NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 50 mM Tris-HCl (pH 7.4), 10% Glycerol) with protease inhibitorcocktail (Roche, Basel, Switzerland) and phosphatase inhibitors (NA₃VO₄1 mM, NaF 100 mM). Then, protein concentration was measured using theBicinchoninic acid (BCA) assay kit (Thermo Scientific, Waltham, Mass.).Whole cell or nuclear lysates were transferred onto polyvinylidenefluoride membranes (Merck Millipore, Billerica, Mass., USA). Afterblocking in 5% non-fat milk (Bio-Rad, Hercules, Calif., USA) for 1 h,the membranes were incubated overnight at 4° C. with primary antibodydiluted in TBST containing 100 mM Tris-HCl (pH 7.5), 1.5 M NaCl, and0.5% Tween-20 obtained from Sigma-Aldrich, to which 5% BSA and 0.1%sodium azide (Sigma-Aldrich) had been added. Specific primary antibodiesto HIF-1α (1:1000), HIF-2α (1:1000), LDH-A (1:1000), LDH-B (1:1000),TLR-4 (1:1000), NF-κB (1:1000), VEGF (1:1000), α-tubulin (1:1000), Actin(1:1000), and GAPDH (1:5000). The next day, the membranes were washedwith TBST and incubated for 2 h with anti-rabbit secondary antibody(1:10000). Immunoblots were developed using a western blot detectionreagent (Abclone, Seoul, Korea) and exposed to x-ray film (Agfa,Leverkusen, Germany) according to the manufacturer's protocol.

Example 12. Immunocytochemistry

ARPE-19 and SK-N-SH cells were fixed on bio-coated coverslips (BDbioscience, San Jose, Calif., USA) by 4% PFA for 20 min and thenincubated with the primary antibodies for 15 h. The primary antibodieswere as follows: HIF-1α (1:300, BD Biosciences, San Jose, Calif., USA);LDH-A (1:300, Santa Cruz Biotechnology, Santa Cruz, Calif., USA); LDH-B(1:300, Santa Cruz Biotechnology, Santa Cruz, Calif., USA); NF-κB(1:300, Santa Cruz Biotechnology, Santa Cruz, Calif., USA); TLR-4(1:300, Santa Cruz Biotechnology, Santa Cruz, Calif., USA); apoE (1:200,Santa Cruz Biotechnology, Santa Cruz, Calif., USA). After PBS wash, thecells were incubated with anti-mouse secondary biotinylated antibody(1:2000, Vector Laboratorie, Burlingame, Calif., USA) and visualizedwith streptavidin conjugated to Fluorescein (Vector Laboratorie,Burlingame, Calif., USA). Coverslips were mounted on microscope slideswith VECTASHIELD® Hard Set™ Mounting Medium with DAPI (VectorLaboratories, Burlingame, Calif., USA). Confocal fluorescence imageswere obtained with confocal laser scanning microscopy (LSCM, Nikon A1+,Tokyo, Japan) under a 60× oil immersion lens.

Example 13. Animal Model for NASH

All experiments were performed under the institutional guidelinesestablished by the Institutional Animal Care and Use Committee at GachonUniversity (IACUC-2017-0008). Female 5-week-old C57BL/6 mice werepurchased form KOATEC (Pyeongtaek, South Korea) and were fedMethionine-Choline diet (MCD) for 4 weeks. When the mice weighed 15 geach, the mice were autopsied randomly to identify nonalcoholicsteatohepatitis (NASH). The mice were injected twice daily (B.I.D) withsubcutaneous calcium lactate (2 mg/kg) for 4 weeks.

Example 14. Animal Model for Age-Related Macular Degeneration

All experiments were performed under the institutional guidelinesestablished by the Institutional Animal Care and Use Committee at GachonUniversity (IACUC-2017-0008). Six-week-old female C57BL/6 mice (20-25 g)were purchased from Orient (Charles River Korea, Seoul, Korea). Allanimals were maintained in a 12-hour light/dark cycle (light on, 08:00)at 22° C.−25° C. with free access to food and water. The mice wereplaced individually in an induction chamber, and anesthesia was inducedwith 2% isoflurane (HANA PHARM CO., Seoul, Korea). The lesions wereinduced in each eye of the mice by laser photocoagulation (SDL405-700,SD Laser, China). The laser pulses were from blue laser (wavelength, 405nm; SD Laser). Laser parameters were 100 μm spot size, 700 mw power and1 s exposure time. A total of 15 mice were randomized into three groups:(i) not subjected to laser induction (n=3); (ii) subjected to laserburns and injected with the vehicle buffer (n=5); and (iii) CaLa, 2mg/kg/day (B.I.D) (n=7). CaLa treatment was started the days after laserphotocoagulation and was treated subcutaneously for 14 days. (33, 34)

Example 15. Histological Analysis for NASH Model

Mouse liver tissues (n=5) were dissected and fixed in 4%paraformaldehyde PBS solution for 15 h at 4° C. Fixed liver tissues wereembedded in paraffin and sectioned at 10 μm. Slides were incubated at55° C. for 2 h, and subsequently deparaffinized in xylene and rehydratedin a graded ethanol series and used for hematoxylin (Merck, Darmstadt,Germany) and eosin (H&E) staining (35). Paraffin sections (10 μm thick)stained with H&E were imaged using a Leica DM 1000 LED microscope (LeicaMicrosystems, Wetzlar, Germany).

Example 16. Histological Analysis for Age-Related Macular Degeneration

The histopathologic examination of retinal lesions was performed 2 weeksafter 2 mg/kg of CaLa was administered subcutaneously. The enucleatedeyes were fixed with 4% paraformaldehyde (PFA) for 24 h at 4° C., andeyes obtained by removing the anterior segments were washed three timesin phosphate-buffered saline. The fixed flat-mounts were dehydrated in agraded series of ethanol and embedded in paraffin. Paraffin sections (10μm thick) were stained with hematoxylin (Merck, Darmstadt, Germany) andeosin (Sigma-Aldrich, St Louis, Mo., USA) (H&E) and were imaged using aLeica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany). Thechoroidal lesion and neovascularization on the flat-mounts were observedby confocal microscopy (Carl Zeiss, Oberkochen, Germany).

Example 17. Masson Trichrome Stain

Mouse liver tissues (n=5) were dissected and fixed in 4%paraformaldehyde PBS solution for 15 h at 4° C. Fixed tissues wereembedded in paraffin and sectioned at 5 mm. Slides were incubated at 55°C. for 2 h, and subsequently deparaffinized in xylene and rehydrated ina graded ethanol series and re-fix in Bouin's solution for 1 hr at 56°C., remove the yellow color to rinse running tap water for 5-10 min andstained Biebrich scarlet-acid fuchsin solution for 5 min then inphosphomolybdic-phosphotungstic acid solution (ratio 1:1) for 30minutes, and aniline blue solution and stain for 15 minutes. Rinsebriefly in distilled water and in 1% acetic acid solution for 2-5 min.Dehydrate 95% ethyl alcohol, absolute ethyl alcohol and clear in xylene.Mount with mounting medium. Imaged using a Leica DM 1000 LED microscope(Leica Microsystems, Wetzlar, Germany).

Example 18. Results

FIG. 1 is an in vitro experiment of human leukemic monocytes. Westernblot results show that inflammatory factor (NF-κb) was decreased by 2.5mM calcium lactate treatment in THP-1 cells (human leukemic monocyte)hypoxia condition.

FIG. 2 is an in vitro experiment of human leukemic monocytesdifferentiated into macrophages. Western blot results show thatinflammatory factor (NF-κb) was decreased by 2.5 mM calcium lactatetreatment in differentiated THP-1 cells with PMA(phorbol-12-myristate-13-acetate) (M0 macrophage) under normoxia andhypoxia conditions.

FIG. 3 provides Western blot results showing that inflammatory factor(NF-κb) was decreased by 2.5 mM calcium lactate treatment indifferentiated THP-1 cells with interferon gamma (IFN-γ) andlipopolysaccharide (LPS) (M1 macrophage) under normoxia and hypoxiaconditions.

FIG. 4 provides Western blot results showing that inflammatory factor(NF-κb) was decreased by 2.5 mM calcium lactate treatment indifferentiated THP-1 cells with interleukin-4 (IL-4) and interleukin-10(IL-10) (M2 macrophage) under normoxia and hypoxia conditions.

FIG. 5 is a liver fibrosis in vitro experiment. Western blot resultsshow that hypoxia inducible factor-la (HIF-1α) was decreased by 2.5 mMcalcium lactate treatment in LX-2 cells (human hepatic stellate cell)under hypoxia condition.

FIG. 6 is a human endothelial cell in vitro experiment. Western blotresults show that hypoxia-mediated factors (lactate dehydrogenase A)were decreased by 2.5 mM calcium lactate treatment in human umbilicalvein endothelial cells under hypoxia condition.

FIG. 7 is a human fibroblast in vitro experiment. Western blot resultsshow that lactate dehydrogenase B was increased by 2.5 mM calciumlactate treatment in CCD-18Co cells (human colon fibroblast) underhypoxia condition.

FIG. 8 is a brain CID in vitro experiment. Western blot results showthat hypoxia-mediated factors were decreased by 2.5 mM calcium lactatetreatment in SK-N-SH cells (brain epithelium) under normoxia and hypoxiaconditions.

FIG. 9 is a liver CID in vitro experiment. Western blot results showthat hypoxia-mediated factors were changed by 2.5 mM calcium lactatetreatment in HepG2 cells (liver epithelium) under hypoxia condition.

FIG. 10 provides Western blot results showing that inflammatory factor(TLR-4) was decreased by 2.5 mM calcium lactate treatment in HepG2 cells(liver epithelium) under hypoxia condition.

FIG. 11 is an eye CID in vitro experiment. Western blot results showthat hypoxia-mediated factors were decreased by 2.5 mM calcium lactatetreatment in ARPE-19 cells (retinal pigment epithelium) under normoxiaand hypoxia conditions.

FIG. 12 provides immunocytochemistry results showing that hypoxiainducible factor-la (HIF-1α) was decreased by 2.5 mM calcium lactatetreatment in ARPE-19 cells (retinal pigment epithelium) under hypoxiacondition.

FIG. 13 provides immunocytochemical results showing that lactatedehydrogenase A was decreased by 2.5 mM calcium lactate treatment in theARPE-19 cells (retinal pigment epithelium) under normoxia condition.

FIG. 14 provides immunocytochemical results showing that inflammatoryfactor (nuclear factor-kappa B) was decreased by 2.5 mM calcium lactatetreatment in the ARPE-19 cells (retinal pigment epithelium) underhypoxia condition.

FIG. 15 provides immunocytochemical results showing that inflammatoryfactor (toll like receptor 4) was decreased by 2.5 mM calcium lactatetreatment in the ARPE-19 cells (retinal pigment epithelium) underhypoxia condition.

FIG. 16 provides immunocytochemical results showing that lactatedehydrogenase B was increased by 2.5 mM calcium lactate treatment in theARPE-19 cells (retinal pigment epithelium) under hypoxia condition.

FIG. 17 provides immunocytochemical results showing that drusen marker(Apolipoprotein E) was decreased by 2.5 mM calcium lactate treatment inthe ARPE-19 cells (retinal pigment epithelium) under hypoxia condition.

FIG. 18 provides Western blot results showing that inflammatory factor(NF-κb) was decreased by 2.5 mM calcium lactate treatment in ARPE-19cells (retinal pigment epithelium) under hypoxia condition.

FIG. 19 shows that there is no toxicity in ARPE-19 cells (retinalpigment epithelium) by 2.5 mM calcium lactate treatment. Calcium lactateonly plays an important role in the metabolic change of injuredepithelial cells.

FIG. 25 provides Western blot results showing that inflammatory factor(NF-κb) was decreased by 2.5 mM calcium lactate treatment in the tissueof retinal pigment epithelium.

Example 19. Targeting Digestive Disease (NFALD, NASH)

Materials and Methods

Animal Model for liver diseases. All experiments were performed underthe institutional guidelines established by the Institutional AnimalCare and Use Committee at Gachon University (IACUC-2017-0008). Female5-week-old C57BL/6 mice were purchased form KOATEC (Pyeongtaek, SouthKorea) and were fed Methionine-Choline (MCD) diet for 4 weeks. The micewere injected twice daily (B.I.D) with subcutaneous calcium lactate (2mg/kg) for 4 weeks. Histological analysis. Mouse Liver tissues (n=5)were dissected and fixed in 4% paraformaldehyde PBS solution for 15 h at4° C. Fixed liver tissues were embedded in paraffin and sectioned at 10Slides were incubated at 55° C. for 2 h, and subsequently deparaffinizedin xylene and rehydrated in a graded ethanol series and used forhematoxylin (Merck, Darmstadt, Germany) and eosin (H&E) staining.Paraffin sections (10 μm thick) stained with H&E were imaged using aLeica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany

Masson Trichrome Stain. Mouse Liver tissues (n=5) were dissected andfixed in 4% paraformaldehyde PBS solution for 15 h at 4° C. Fixedtissues were embedded in paraffin and sectioned at 5 mm. Slides wereincubated at 55° C. for 2 h, and subsequently deparaffinized in xyleneand rehydrated in a graded ethanol series and re-fix in Bouin's solutionfor 1 hr at 56° C., remove the yellow color to rinse running tap waterat 5˜10 min and stained Biebrich scarlet-acid fuchsin solution for 5 minthan in phosphomolybdic-phosphotungstic acid solution (ratio 1:1) for 30minutes, and aniline blue solution and stain for 15 minutes. Rinsebriefly in distilled water and in 1% acetic acid solution for 2-5minutes. Dehydrate 95% ethyl alcohol, absolute ethyl alcohol and clearin xylene. Mount with mounting medium. Imaged using a Leica DM 1000 LEDmicroscope (Leica Microsystems, Wetzlar, Germany).

FIG. 20 is a liver CID (NASH) in vivo experiment. To induce NASH, micewere fed methionine-choline deficient (MCD) diet with polyunsaturatedfat for 8 weeks. The blood chemistry results show that serum AST and ALTwere significantly decreased by 2 mg/kg calcium lactate treatment.**P<0.001 vs. methionine-choline deficient (MCD). Results are MEAN±SD.

FIG. 21 provides immunohistochemical results showing that lipid droplets(white pores in liver tissues) were not observed in the calciumlactate-treated group while the lipid droplets were well observed in themethionine-choline deficient (MCD) group. Wteatosis was prevented by 2mg/kg calcium lactate treatment. The results show that steatosis isprevented by calcium lactate treatment.

FIGS. 22A, 22B, and 22C provide immunohistochemical results showing thatin the liver lipid droplets (white pores in liver tissues) are notpresent in the 2 mg/kg calcium lactate-treated group, similar to thecontrol group, while they are found in the methionine-choline deficient(MCD) group.

FIG. 23 provides immunohistochemical results showing that in the liverimmune cell infiltration is decreased in the 2 mg/kg calciumlactate-treated group, similar to the control group, while immune cellinfiltration is increased in the methionine-choline deficient (MCD)group.

FIG. 24 provides immunohistochemical results showing that in the liverfibrosis (collagen accumulation site; white arrows) is not present inthe 2 mg/kg calcium lactate-treated group, similar to the control group,while it is present in the methionine-choline deficient (MCD) group.

Example 20. Targeting Ocular Disease (Age-Related Macular Degeneration(AMD), Keratitis, and Diabetic Retinopathy)

Materials and Methods

Animals. All experiments were performed under the institutionalguidelines established by the Institutional Animal Care and UseCommittee at Gachon University (IACUC-2017-0008). Six-week-old C57BLmice were purchased from Orient (Charles River Korea, Seoul, Korea). Allanimals were maintained in a 12-hour light/dark cycle (light on, 08:00)at 22-25° C. with free access to food and water.

Preparation of calcium lactate. Calcium lactate was purchased from SigmaAldrich (St. Louis, Mo., USA). Two mg/kg of calcium lactate wasdissolved in saline for daily subcutaneous injection (21 days).

Animal model for ocular disease. The mice were placed individually in aninduction chamber, and anesthesia was induced with 2% isoflurane (HANAPHARM CO., Seoul, Korea). The laser-induced rupture of Bruch's membranewas induced in both eyes of mice by laser photocoagulation (SDL405-700,SD Laser, China). The laser pulses were from green laser (wave length,532 nm; Visulas, 532; SD Laser). Laser parameters were 100 μm spot size,700 mw power and 1 s exposure time.

Preparation of choroidal flat mounts. Twenty one days after laserinjury, mice in each group were anesthetized and perfused through theleft ventricle with 1.0 mL PBS containing 25 mg fluoresceinisothiocyanate-dextran (Sigma-Aldrich, St. Louis, Mo., USA). Eyes wereenucleated and fixed in 4% paraformaldehyde for 1 hour. Retinal pigmentepithelium-choroid-sclera eyecups were prepared after hemisecting theeye, with total removal of the lens, vitreous body, and retina. Retinalpigment epithelium-choroid-sclera eyecups were flattened by the creationof four or five radial incisions, from the edge to the equator, andflat-mounted in aqua-mount.

Histological analysis. The fixed flat-mounts were dehydrated in a gradedseries of ethanol and embedded in paraffin. Paraffin sections (10 μmthick) were stained with hematoxylin (Merck, Darmstadt, Germany) andeosin (Sigma-Aldrich, St Louis, Mo., USA) (H&E) and were imaged using aLeica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany). Thechoroidal lesion and neovascularization on the flat-mounts were observedby confocal microscopy (Carl Zeiss, Oberkochen, Germany).

Results

FIG. 26 provides representative images for H&E staining of lesion ofinner layer of choroidal flat-mount. An abnormal morphology of retinalpigment epithelium was observed after AMD induction (middle image). Themorphology of the retinal pigment epithelium was recovered in a similarmanner to the normal control epithelium (left image) by treatment with2.5 mM calcium lactate (right image).

FIG. 27 provides fluorescence images for lesion of inner layer ofchoroid on flat-mount by laser-induced AMD. The choroidal lesion wasobserved after AMD induction (middle image). The choroidal lesion wasrestored after treatment with calcium lactate (right image).

FIG. 28 provides fluorescence images for lesion of inner layer andneovascularization of choroid on flat-mount by laser-induced AMD. Thechoroidal lesion and neovascularization were observed after AMDinduction (middle image). The choroidal lesion and neovascularizationwere restored after treatment with calcium lactate (right image), samewith the normal control (left image).

Example 21. Targeting Alzheimer & Stroke

Materials and Methods

Animals. All experiments were carried out in accordance with animal careguidelines under Korean law (11737, rev′d Apr. 5, 2013) and theguidelines set by the Institutional Animal Care and Use Committee atGachon university (LCDI-2018-0073). Eight-week old male Sprague-Dawley(SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic ofKorea) and bred under specific-pathogen-free (SPF) conditions. Allanimals were maintained in a 12-hour light/dark cycle at 22-25° C. withfree access to food and water.

Preparation of Lipopolysaccharides (LPS) and Calcium Lactate. Dissolve 1mg of LPS powder in 1 ml of saline. Keep the tubes on ice if LPS isgoing to be used fresh. For long storage, screw cap vials are preferred.The aliquots were stored at −20° C. Calcium lactate was purchased fromSigma Aldrich (St. Louis, Mo., USA). Two mg/kg of calcium lactate weredissolved in saline for daily subcutaneous injection (21 days).

Intracranial injection of LPS. The animals were placed in a smallacrylic cage that was coupled with an anesthesia machine that allowedthem to breathe freely under gaseous anesthesia (2% isoflurane,Spartanburg, S.C., USA). After full anesthesia of an animal wasachieved, it was positioned on an operating table (while maintaining thegaseous anesthesia) with the superior aspect of the neck hyperextendedto expose the posterior region. The animal fur of head was shaved withan electric clipper, and the skin was swabbed with iodine. LPS was keptit on ice before use. All the surgical materials disinfected with 70%ethanol. Hamilton syringe barrel inner space was cleaned with distilledwater and 70% ethanol. The Hamilton syringe was placed in the syringeholder. The animal in the stereotaxic apparatus was placed on horizontalposition. A midline incision (about 1 cm) on the skin of head was madewith the scalpel. The needle of Hamilton syringe was placed on thebregma. Read the three coordinates (AP: −3.6, LM: 2.0, and DV: −3.8) onthe manipulator (in mm) and write them down; this was used for startingpoint. Slowly, set the DV coordinate of the injection point; thisinserts the needle tip into the brain until the injection point. The LPSsolution (2 μl) was released at an approximate flow rate of 0.5 μl/min.After the injection, the needle was removed very slowly. The wound areawas closed by suturing, and then disinfectant was applied. The rat wasmarked for further identification.

Immunohistochemistry. Brain tissue was sectioned at 5 μm on cryostat andstained for histological examination. Microscopic images were obtainedat 200× magnification. For immunohistochemistry, sections were blockedwith 1% BSA and 10% NGS in 0.05 M PBS for 1 h at room temperature andthen reacted with rabbit anti-Iba-1 (1:1000, Bioss, MA, USA) at 4° C.two overnights. ABC staining system was used to visualize microglia(Iba-1) in the sections (Vector Laboratories, CA, USA).

Results

FIG. 29 provides an experimental scheme for establishing Alzheimer andstroke diseases. OMT-110 is calcium lactate. FIG. 30 providesrepresentative images for showing brain damage by LPS. Contralateral:normal region. Ipsilateral: LPS injection.

FIG. 31 shows the recovery of damaged brain tissue and the reduction ofmicroglia infiltration that recruited upon brain damage. The resultsshow that the inflammatory reaction, which exacerbates Alzheimer's andstroke, is greatly reduced.

Example 22. Targeting Parkinson's Disease

Materials and Methods

Animals. All experiments were carried out in accordance with animal careguidelines under Korean law (11737, rev′d Apr. 5, 2013) and theguidelines set by the Institutional Animal Care and Use Committee atGachon university (LCDI-2018-0073). Eight-week old male Sprague-Dawley(SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic ofKorea) and bred under specific-pathogen-free (SPF) conditions. Allanimals were maintained in a 12-hour light/dark cycle at 22-25° C. withfree access to food and water.

Preparation of desipramine, 6-OHDA, and calcium lactate. Desipramine(12.5 mg/kg; noradrenaline transporter blocker; Sigma Aldrich, St.Louis, USA) was dissolved in saline for intraperitoneal injection.6-OHDA (20 μg/rat, Sigma Aldrich, St. Louis, USA) was dissolved insaline for intracranial injection. 2 mg/kg of calcium lactate (SigmaAldrich, St. Louis, USA) was dissolved in saline for daily subcutaneousinjection (21 days).

Injection of desipramine and 6-OHDA. The animals were injected withdesipramine into the abdominal cavity (intraperitoneal injection) 30 minbefore 6-OHDA injection. 30 min after Desipramine injection, the animalswere placed in a small acrylic cage that was coupled with an anesthesiamachine that allowed them to breathe freely under gaseous anesthesia (2%isoflurane, Spartanburg, S.C., USA). After full anesthesia of an animalwas achieved, it was positioned on an operating table (while maintainingthe gaseous anesthesia) with the superior aspect of the neckhyperextended to expose the posterior region. The animal fur of head wasshaved with an electric clipper, and the skin was swabbed with iodine.All the surgical materials disinfected with 70% ethanol. Hamiltonsyringe barrel inner space was cleaned with distilled water and 70%ethanol. The Hamilton syringe was placed in the syringe holder. Theanimal in the stereotaxic apparatus was placed on horizontal position. Amidline incision (about 1 cm) on the skin of head was made with thescalpel. The needle of Hamilton syringe was placed on the bregma. Thethree coordinates (AP: −5.6, LM: 2.0, and DV: −7.6) on the manipulator(in mm) were read and used as the starting point. The DV coordinate ofthe injection point was set slowly; this inserts the needle tip into thebrain until the injection point. The 6-OHDA solution (20 μg/4 μl) wasreleased at an approximate flow rate of 1 μl/min. After the injection,the needle was removed very slowly. The wound area was closed bysuturing, and then disinfectant was applied. The rat was marked forfurther identification.

Immunohistochemistry. Brain tissue was sectioned at 5 μm on cryostat andstained for histological examination. Microscopic images were obtainedat 200× magnification. For immunohistochemistry, sections were blockedwith 1% BSA and 10% NGS in 0.05 M PBS for 1 h at room temperature andthen reacted with rabbit anti-tyrosine hydroxylase (1:500, Chemicon,Tokyo, Japan) at 4° C. two overnights. ABC staining system was used tovisualize dopaminergic neuron in the sections (Vector Laboratories, CA,USA).

Results

FIG. 32 provides an experimental scheme for establishing Parkinson'sdisease. OMT-110 is calcium lactate.

FIG. 33 shows the recovery of dopaminergic neuron by treatment withcalcium lactate. Parkinson's disease is a neurodegenerative disorderthat affects dopamine-producing by disrupting dopaminergic neurons in aspecific area of the brain (substantia nigra).

Example 23. Targeting Multiple Sclerosis and Spinal Cord Injury

Materials and Methods

Animals. All experiments were carried out in accordance with animal careguidelines under Korean law (11737, rev′d Apr. 5, 2013) and theguidelines set by the Institutional Animal Care and Use Committee atGachon university (LCDI-2018-0073). Eight-week old male Sprague-Dawley(SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic ofKorea) and bred under specific-pathogen-free (SPF) conditions. Allanimals were maintained in a 12-hour light/dark cycle at 22-25° C. withfree access to food and water.

Preparation of Lipopolysaccharides (LPS) and Calcium Lactate. Dissolve 1mg of LPS powder in 1 ml of saline. Keep the tubes on ice if LPS isgoing to be used fresh. For long storage, use screw cap vials arepreferred. The aliquots were stored at deep freezer (−20° C.). Calciumlactate was purchased from Sigma Aldrich (St. Louis, USA). Two mg/kg ofcalcium lactate was dissolved in saline for daily subcutaneous injection(21 days).

Intraspinal injection of LPS. The animals were placed in a small acryliccage that was coupled with an anesthesia machine that allowed them tobreathe freely under gaseous anesthesia (2% isoflurane, Spartanburg,S.C., USA). After full anesthesia of an animal was achieved, it waspositioned on an operating table (while maintaining the gaseousanesthesia). For intraspinal injection of LPS, the laminectomy site ofrats was exposed. Two small holes, 1 mm apart, were made in the duraover the left dorsal column. A volume of 1 μl of LPS solution wascarefully drawn up into a sterile Hamilton syringe, and the LPS solutionwas slowly injected into the spinal cord. The syringe maintained inplace for an additional 2 min to prevent back-flux from the injectionsite. Then, the injection site was closed by surgical clips.

Immunohistochemistry. Spinal cord tissue was sectioned at 5 μm oncryostat and stained for histological examination. Microscopic imageswere obtained at 200× magnification. For immunohistochemistry, sectionswere blocked with 1% BSA and 10% NGS in 0.05 M PBS for 1 h at roomtemperature and then reacted with rabbit anti-Iba-1 (1:1000, Bioss, MA,USA) at 4° C. two overnights. ABC staining system was used to visualizemicroglia (Iba-1) in the sections (Vector Laboratories, CA, USA).

Results

FIG. 34 provides an experimental scheme for establishing multiplesclerosis. OMT-110 is calcium lactate.

FIG. 35 shows the recovery of demyelinate spinal cord and the reductionof microglia infiltration by treatment with calcium lactate.Demyelination of central nervous system and immune infiltration occur inassociation with inflammation in a number of disorders, includingmultiple sclerosis and spinal cord injury.

Example 24. Targeting Vascular Disease (Atherosclerosis, CerebralHemorrhage, and Myocardial Infarction)

Materials and Methods

Animals. All experiments were carried out in accordance with animal careguidelines under Korean law (11737, rev′d Apr. 5, 2013) and theguidelines set by the Institutional Animal Care and Use Committee atGyeonggi bio center (2017-11-0008, South Korea). Fibe-week old maleBALB/c ApoP^(shi) mice were obtained from Japan SLC Inc. (Shizuoka,Japan) and bred under specific-pathogen-free (SPF) conditions. Allanimals were maintained in a 12-hour light/dark cycle at 22-25° C. withfree access to food and water.

Preparation of Calcium Lactate. Calcium lactate was purchased from SigmaAldrich (St. Louis, Mo., USA). Two mg/kg of calcium lactate wasdissolved in saline for daily subcutaneous injection (21 days).

Animal model for vascular disease. BALB/c ApoE^(shi) species is used forspontaneous induction of vascular disease. In this investigation, a highfat (40%) diet was provided for 4 weeks to induce strongatherosclerosis.

Histological analysis. The thoracic aorta was transected, and a half ofthe aorta was fixed in 10% neutral buffer formalin fixative solution.Then, histological analysis was performed. Histological analysis wasperformed with H&E and Oil red O staining. Aortic wall thickness,atherosclerotic plaque areas, foam cell numbers, and lipidated regionswere analyzed.

Statistical analysis. Statistical analysis was performed using SPSSstatistics, and the results were analyzed using Student t-test.Histologic analysis was performed using the Mann-Whitney method. P<0.05was considered statistically significant.

Results

TABLE 1 Quantitative analysis for histological analysis. All clinicalindicators of arteriosclerosis were significantly increased afterdisease induction. However, the indicators were significantly decreasedafter treatment with calcium lactate. *: significantly different betweenG2 and G1, P < 0.05; ++: significantly different between G2 and G3, P <0.01; +: significantly different between G2 and G3, P < 0.05.HISTOPATHOLOGICAL ANALYSIS MALE MEAN ATHERO- AORTA SCLEROTIC FOAM LIPIDWALL PLAQUE CELL DEPOSITED THICKNESS AREAS NUMBERS REGIONS Group (μm)(mm²) (cells/mm²) (mm²) G1  47.71 ± 2.43 0.00 ± 0.00  0.00 ± 0.00 0.01 ±0.00 G2 147.51 ± 23.58* 0.50 ± 0.08* 135.75 ± 31.76* 0.41 ± 0.06* G3 85.83 ± 8.70⁺ 0.10 ± 0.03⁺⁺  39.00 ± 10.13⁺ 0.09 ± 0.03⁺⁺ Data wereexpressed as mean ± S.E.M. The results were statistically analyzed byMann-Whitney methods. G1: Normal control (Saline); G2: Vehicle control(Saline); G3: Calcium lactate; G2-G3: High cholesterol diet suppliedApoE-deficient mice (Atherosclerosis)

Evidence for direct targeting on Atherosclerosis

TABLE 2 Clinical blood chemistry CLINICAL BLOOD BIOCHEMISTRY (mg/dL)MALE GROUP TCHO TG LDL HDL G1  113.7 ± 3.0 75.7 ± 1.5  15.0 ± 2.9  87.3± 13.0 G2 2092.4 ± 349.2** 48.6 ± 6.8* 1541.6 ± 30.9** 220.4 ± 11.7** G32328.0 ± 56.7 54.8 ± 2.8 1537.1 ± 14.2 197.2 ± 3.7 Data were expressedas mean ± S.E.M. statistically analyzed by student t-test methods.**statistically different between G2 and G1, P < 0.01 *statisticallydifferent between G2 and G1, P < 0.05 G1: Normal control (Saline, n = 3)G2: Vehicle control (Saline, n = 8) G3: Test article (Calcium lactatepentahydrate, n = 8) G2-G3: High cholesterol diet suppliedApoE-deficient mice

Pathogenic factors such as cholesterol (TCHO), LDL cholesterol (LDL),triglycerides (TG), and HDL-cholesterol (HDL) were not controlled bycalcium lactate. The combined form of calcium with lactate acts as asingle ingredient directly on the disease site (see FIG. 36).

FIG. 36 provides representative histological profiles of the aortatissues. The tissues in left and middle side of pictures are a H&Estaining. The tissues in right side of pictures are an oil red stainingfor distinguishing the atherosclerotic lesions. The atheroscleroticlesions were decreased by treatment with calcium lactate.

Example 25. Targeting Periodontal Disease (Pulpitis and Periodontitis)

Materials and Methods

Animals. All experiments were carried out in accordance with animal careguidelines under Korean law (11737, revised Apr. 5, 2013) and theguidelines set by the Institutional Animal Care and Use Committee atGachon university (LCDI-2018-0073). Eight-week old male Sprague-Dawley(SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic ofKorea) and bred under specific-pathogen-free (SPF) conditions. Allanimals were maintained in a 12-hour light/dark cycle at 22-25° C. withfree access to food and water.

Preparation of Lipopolysaccharides (LPS) and Calcium Lactate. Dissolve 1mg of LPS powder in 100 μl of saline. Keep the tubes on ice if LPS isgoing to be used fresh. For long storage, use screw cap vials arepreferred. The aliquots were stored in a deep freezer (−20° C.). Calciumlactate was purchased from Sigma Aldrich (St. Louis, Mo., USA). Twomg/kg of calcium lactate was dissolved in saline for daily subcutaneousinjection (21 days).

Animal model for periodontal disease. Periodontal disease was induced byintragingival injection of LPS (1 mg/100 μl) in sterile saline. A finehypodermic needle was inserted at the mesiolateral aspect of the firstright mandibular molar and the tip moved distally so that the injectionwas made at the interdental papilla between the first and second molars.The injection was slowly made, and the needle held in place for 10seconds post injection to ensure that the LPS was not lost through theneedle track.

Histological analysis. Rat gingiva including tooth were dissected andfixed in 4% paraformaldehyde for 2 days at 4° C. Fixed tissues wereembedded in paraffin and sectioned at 10 μm. Slides were incubated at55° C. for 2 h, and subsequently deparaffinized in xylene and rehydratedin a graded ethanol series and re-fix in Bouin's solution for 1 h,remove the yellow color to rinse running tap water for 5˜10 min andstained with Biebrich scarlet-acid fuchsin solution for 5 min than inphosphomolybdic-phosphotungstic acid solution (ratio 1:1) for 30 min,and aniline blue solution for 15 min. Rinse briefly in distilled waterand in 1% acetic acid solution for 5 min. Dehydrate 95% ethyl alcohol,absolute ethyl alcohol and clear in xylene. Mount with mounting medium.The stained tissues were Imaged using a Leica DM 1000 LED microscope(Leica Microsystems, Wetzlar, Germany).

Results

FIG. 37 shows Masson's trichrome staining for periodontal tissues. LPStreatment induces periodontal disease, which causes loss of ligamentsand gingiva around the root of tooth (middle image). The ligaments andgingiva were recovered after treatment with calcium lactate (rightimage), same with the normal control (left image).

FIG. 38 shows Masson's trichrome staining for the upper gum. The uppergum around the periodontal tissue that was treated with LPS showsabnormal morphology by severe inflammation (middle image). Themorphology of the upper gum was recovered after treatment with calciumlactate (right image), same with the normal control (left image).

Example 26. Targeting Musculoskeletal Disease (Rheumatoid Arthritis)

Materials and Methods

Animals. All experiments were carried out in accordance with animal careguidelines under Korean law (11737, rev′d Apr. 5, 2013) and theguidelines set by the Institutional Animal Care and Use Committee atGyeonggi bio center (2017-11-0003, South Korea). Six-week old maleDBA1/J mice were obtained from Japan SLC Inc. (Shizuoka, Japan) and bredunder specific-pathogen-free (SPF) conditions. All animals weremaintained in a 12-hour light/dark cycle at 22-25° C. with free accessto food and water.

Preparation of agents. Two mg/mL Type II collagen was mixed with thesame volume of complete Freund's adjuvant (CFA) for primary induction ofrheumatoid arthritis. Two mg/mL Type II collagen was mixed with the samevolume of incomplete Freund's adjuvant (IFA) for secondary induction ofrheumatoid arthritis. Two mg/kg of calcium lactate was dissolved insaline for daily subcutaneous injection (21 days).

Animal Model for Rheumatoid Arthritis

Primary induction: 0.1 mL of the mixed solution (Type II collagen+CFA)was injected subcutaneously into the area of 1.5 cm from the base oftail of mice. The administration site and depth were the same in allmice.

Secondary induction: 21 days after primary induction, 0.1 mL of themixed solution (Type II collagen+IFA) was injected subcutaneously intothe area of 1.5 cm from the base of tail of mice. The administrationsite and depth were the same in all mice.

Histological analysis. The mice foot and knee joint were dissected andfixed (4% paraformaldehyde) and decalcified for 2 days at 4° C. Thetissues were embedded in paraffin and sectioned at 15 μm. Slides wereincubated at 55° C. for 2 h, and subsequently deparaffinized in xyleneand rehydrated in a graded ethanol series and stained with Biebrichscarlet-acid fuchsin solution for 5 min than inphosphomolybdic-phosphotungstic acid solution (ratio 1:1) for 30 min,and aniline blue solution for 15 min. Rinse briefly in distilled waterand in 1% acetic acid solution for 5 min. Dehydrate 95% ethyl alcohol,absolute ethyl alcohol and clear in xylene. Mount with mounting medium.The stained tissues were Imaged using a Leica DM 1000 LED microscope(Leica Microsystems, Wetzlar, Germany).

Results

FIG. 39 shows Masson's trichrome staining for foot and knee jointtissues. Inflammatory edema was observed in the toes and knee joint byrheumatoid arthritis (middle images). 21 days after treatment with CaLa,the inflammatory edema clearly decreased in the toes and knee joint(lower images), and it was morphologically recovered same with thenormal control (upper images).

Example 27. Targeting Digestive Disease (Inflammatory Bowel Disease:Crohn's and Colitis)

Materials and Methods

Animals. All experiments were carried out in accordance with animal careguidelines under Korean law (11737, rev′d Apr. 5, 2013) and theguidelines set by the Institutional Animal Care and Use Committee atGachon university (LCDI-2018-0073). Six-week old male Sprague-Dawley(SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic ofKorea) and bred under specific-pathogen-free (SPF) conditions. Allanimals were maintained in a 12-hour light/dark cycle at 22-25° C. withfree access to food and water.

Agents and animal model. To induce Crohn's disease and colitis,Indomethacin was purchased from Sigma Chemical (St. Louis, Mo., USA) andwas dissolved in saline. Two mg/kg of calcium lactate was dissolved insaline for daily subcutaneous injection (21 days). Two models ofinflammatory bowel disease were used. Crohn's disease was induced byoral administration of Indomethacin (7 mg/kg), and colitis was inducedby subcutaneous injections of Indomethacin (7 mg/kg) daily at a 24 hintervals (FIGS. 40 and 42).

Histological analysis. The fixed flat-mounts were dehydrated in a gradedseries of ethanol and embedded in paraffin. Paraffin sections (5 μmthick) were stained with hematoxylin (Merck, Darmstadt, Germany) andeosin (Sigma-Aldrich, St Louis, Mo., USA) (H&E) and were imaged using aLeica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

Results

FIG. 40 provides an experimental scheme for inducing Crohn's disease.

FIG. 41 provides representative images for H&E staining of Crohn'sdisease. Transmural inflammation and linear ulceration in intestine wereobserved after induction of Crohn's disease (middle image). Four-weekafter treatment with CaLa, the transmural inflammation and linearulceration were clearly decreased in the intestinal tissues (lowerimages), and it was morphologically recovered same with the normalcontrol (upper images).

FIG. 42 provides an experimental scheme for inducing colitis.

FIG. 43 provides representative images for H&E staining of colitis.Superficial ulceration, polyp, and inflammation in intestine wereobserved after induction of colitis (middle image). Four-week aftertreatment with CaLa, the Superficial ulceration, polyp, and inflammationwere clearly decreased in the intestinal tissues (lower images), and itwas morphologically recovered same with the normal control (upperimages).

Some additional embodiments disclosed herein include but are not limitedto the following:

-   -   A. A method of treating asthma, pulmonary fibrosis, obesity,        gastroenteritis, chronic inflammatory bowel disease, and/or        atopic dermatitis, the method comprising administering to a        subject in need thereof a therapeutically effective amount of        calcium lactate.    -   B. A method of treating chronic obstructive pulmonary disease,        pneumonitis, keratitis, atherosclerosis, arteriosclerosis,        myocarditis, diabetes, rheumatoid arthritis, pulpitis,        periodontitis, and/or psoriasis, the method comprising        administering to a subject in need thereof a therapeutically        effective amount of calcium lactate.    -   C. A method of treating Alzheimer's disease, stroke, Parkinson's        disease, multiple sclerosis, age-related macular degeneration,        non-alcoholic fatty liver disease, sepsis, and/or osteoporosis,        the method comprising administering to a subject in need thereof        a therapeutically effective amount of calcium lactate.    -   D. The method of any one of embodiments A-C, wherein the calcium        lactate is administered in a pharmaceutical composition        comprising a pharmaceutically acceptable carrier, excipient, or        diluent.    -   E. The method of any one of embodiments A-D, wherein the        pharmaceutical composition is formulated into liquid, powder,        aerosol, injection, fluid transfusion, patch, capsule, pill,        tablet, depot, or suppository.    -   F. The method of any one of embodiments A-D, wherein the        pharmaceutical composition comprises a therapeutically effective        amount of calcium lactate as an active agent and a        pharmaceutically acceptable polysaccharide, polymer, lipid, or        combinations thereof.    -   G. The method of embodiment F, wherein the pharmaceutical        composition comprises the calcium lactate and the        polysaccharide.    -   H. The method of embodiment G, wherein a weight ratio of the        calcium lactate to the polysaccharide is 1:<0.2 to 1:5.    -   I. The method of embodiment G, wherein a weight ratio of the        calcium lactate to the polysaccharide is 1:<0.2.    -   J. The method of embodiment G, wherein a weight ratio of the        calcium lactate to the polysaccharide is 1:0.2 to 1:5.    -   K. The method of any one of embodiment F-J, further comprising a        polymer and/or lipid.    -   L. The method of embodiment K, further comprising the polymer        and lipid, wherein the weight ratio of the polymer to the lipid        is 1:0.1 to 1:50.    -   M. The method of any one of embodiments K-L, wherein the weight        ratio of the calcium lactate to the polymer and/or lipid is at        least 1:5.    -   N. The method of embodiment M, wherein a weight ratio of the        calcium lactate to the polymer and/or lipid is 1:5 to 1:30.    -   O. The method of embodiment F, comprising the calcium lactate        and the polymer and/or lipid.    -   P. The method of embodiment O, comprising the polymer and lipid,        wherein the weight ratio of the polymer to the lipid is 1:0.1 to        1:50.    -   Q. The method of any one of embodiments O-P, wherein a weight        ratio of the calcium lactate to the polymer and/or lipid is at        least 1:5.    -   R. The method of any one of embodiments O-Q, wherein the weight        ratio of the calcium lactate to the polymer and/or lipid is 1:5        to 1:30.    -   S. The method of any one of embodiments O-R, wherein the        composition is short-acting.    -   T. The method of any one of embodiments O-R, wherein the        composition is long-acting.    -   U. The method of any one of embodiments F-T, wherein the        composition is an injectable composition.    -   V. The method of any one of embodiments F-U, comprising the        polysaccharide that is a cellulose derivative, pectin,        hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen,        alginate, alginic acid or combinations thereof.    -   W. The method of any one of embodiments F-V, comprising the        polymer that is a poloxamer, polyvinylpyrrolidone, polyethylene        glycol (PEG), polyglycolic lactic acid (PLGA), or combinations        thereof.    -   X. The method of any one of embodiments F-W, comprising the        lipid that is a mono- or tri-fatty acid glycerin ester or        polyethylene glycol, polyethylene glycol esters of vegetable        oils, fatty acid propylene glycol esters, sesame oil, soybean        oil, castor oil, corn oil, palm oil, peanut oil, cacao oil,        cottonseed oil, sunflower seed oil, safflower oil, almond oil,        olive oil, hydrogenated oil, oleic acid, linolenic acid,        linoleic acid, palmitic acid, palmitoleic acid, arachadonic        acid, myristic acid, capric acid, caprylic acid, lauric acid,        stearic acid, ethyl oleate, isopropyl palmitate, octyldodecyl        myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl        alcohol, stearyl alcohol, or combinations thereof.    -   Y. The method of any one of embodiments F-S and U-X, wherein        upon placement of the composition in an in vitro dissolution        test comprising an elution test method at 300 rpm in 200 ml        aqueous medium having a pH of 6.8 at 37° C. using a nylon filter        having a pore size of 45 μm, at least about 40% of the active        agent is released after 6 hours.    -   Z. The method of any one of embodiments F-S and U-Y, wherein        upon placement of the composition in an in vitro dissolution        test comprising an elution test method at 300 rpm in 200 ml        aqueous medium having a pH of 6.8 at 37° C. using a nylon filter        having a pore size of 45 μm, at least about 60% of the active        agent is released after 12 hours.    -   AA. The method of any one of embodiments F-S and U-Z, wherein        upon placement of the composition in an in vitro dissolution        test comprising an elution test method at 300 rpm in 200 ml        aqueous medium having a pH of 6.8 at 37° C. using a nylon filter        having a pore size of 45 μm, at least about 80% of the active        agent is released after 24 hours.    -   BB. The method of any one of embodiments F-S and U-AA, wherein        upon placement of the composition in an in vitro dissolution        test comprising an elution test method at 300 rpm in 200 ml        aqueous medium having a pH of 6.8 at 37° C. using a nylon filter        having a pore size of 45 μm, at least about 90% of the active        agent is released after 48 hours.    -   CC. The method of any one of embodiments F-H, J-R, and T-X,        wherein upon placement of the composition in an in vitro        dissolution test comprising an elution test method at 300 rpm in        200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylon        filter having a pore size of 45 μm, less than about 40% of the        active agent is released after 24 hours.    -   DD. The method of any one of embodiments F-H, J-R, and T-CC,        wherein upon placement of the composition in an in vitro        dissolution test comprising an elution test method at 300 rpm in        200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylon        filter having a pore size of 45 μm, less than about 60% of the        active agent is released after 48 hours.    -   EE. The method of any one of embodiments F-H, J-R, and CC-DD,        wherein upon placement of the composition in an in vitro        dissolution test comprising an elution test method at 300 rpm in        200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylon        filter having a pore size of 45 μm, less than about 80% of the        active agent is released after 72 hours.    -   FF. The method of any one of embodiments F-H, J-R, and CC-EE,        wherein upon placement of the composition in an in vitro        dissolution test comprising an elution test method at 300 rpm in        200 ml aqueous medium having a pH of 6.8 at 37° C. using a nylon        filter having a pore size of 45 μm, less than about 90% of the        active agent is released after 144 hours.    -   GG. The method of any one of embodiments F-FF, wherein the        composition is contained in a sterile glass or polyolefin        container.    -   HH. The method of any one of embodiments A-D, wherein the        calcium lactate is coated with a pharmaceutically acceptable        enteric coating.    -   II. The method of embodiment HH, wherein the enteric coating        comprises hydroxypropyl methyl cellulose phthalate (HPMCP),        cellulose acetate phthalate (CAP), polyvinyl acetate phthalate        (PVAP), shellac, polymer of methacrylic acid and an ester        thereof, or combinations thereof.    -   JJ. The method of any one of embodiments wherein the weight        ratio of the calcium lactate to the enteric coating is 10:0.5 to        1:1.5.    -   KK. The method of any one of embodiments HH-JJ, wherein in an in        vitro dissolution test comprising a USP Paddle method at a        paddle speed of 50 rpm at 37° C., when the composition is placed        in 0.1 HCl for 120 minutes followed by adjusting to pH 6.8 with        phosphate buffer, less than about 20% of the active agent is        released after 30 minutes.    -   LL. The method of any one of embodiments HH-KK, wherein in an in        vitro dissolution test comprising a USP Paddle method at a        paddle speed of 50 rpm at 37° C., when the composition is placed        in 0.1 N HCl for 120 minutes followed by adjusting to pH 6.8        with phosphate buffer, less than 30% of the active agent is        released after 60 minutes.    -   MM. The method of any one of embodiments HH-LL, wherein in an in        vitro dissolution test comprising a USP Paddle method at a        paddle speed of 50 rpm at 37° C., when the composition is placed        in 0.1 N HCl for 120 minutes followed by adjusting to pH 6.8        with phosphate buffer, less than 50% of the active agent is        released after 120 minutes.    -   NN. The method of any one of embodiments HH-MM, wherein in an in        vitro dissolution test comprising a USP Paddle method at a        paddle speed of 50 rpm at 37° C., when the composition is placed        in 0.1 N HCl for 120 minutes followed by adjusting to pH 6.8        with phosphate buffer, less than 10% of the active agent is        released after 120 minutes.    -   OO. The method of any one of embodiments A-B, wherein the        calcium lactate is provided in a food or nutrient composition        comprising calcium lactate.    -   PP. The method of embodiment OO, wherein the composition is an        injectable nutritional supplement.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications, without departing from the general concept of theinvention. Therefore, such adaptations and modifications are intended tobe within the meaning and range of equivalents of the disclosedembodiments, based on the teaching and guidance presented herein. It isto be understood that the phraseology or terminology herein is for thepurpose of description and not of limitation, such that the terminologyor phraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

All of the various aspects, embodiments, and options described hereincan be combined in any and all variations.

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All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

The invention claimed is:
 1. A method of treating inflammatory diseasecomprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a therapeutically effective amount of calciumlactate, and a polymer and/or lipid, wherein the inflammatory disease isselected from the group consisting of Alzheimer's disease, Parkinson'sdisease, atherosclerosis, and inflammatory bowel disease.
 2. The methodof claim 1, wherein the inflammatory bowel disease is Crohn's orcolitis.
 3. The method of claim 1, wherein the pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrier,excipient, or diluent.
 4. The method of claim 1, wherein thepharmaceutical composition is a liquid, powder, aerosol, injection,fluid transfusion, patch, capsule, pill, tablet, depot, or suppository.5. The method of claim 1, wherein the pharmaceutical composition furthercomprises a pharmaceutically acceptable polysaccharide.
 6. The method ofclaim 5, wherein a weight ratio of the calcium lactate to thepolysaccharide is 1:<0.2 to 1:5.
 7. The method of claim 5, wherein aweight ratio of the calcium lactate to the polysaccharide is 1:<0.2. 8.The method of claim 5, wherein a weight ratio of the calcium lactate tothe polysaccharide is 1:0.2 to 1:5.
 9. The method of claim 1, whereinthe weight ratio of the polymer to the lipid is 1:0.1 to 1:50.
 10. Themethod of claim 1, wherein the weight ratio of the calcium lactate tothe polymer and/or lipid is at least 1:5.
 11. The method of claim 1,wherein a weight ratio of the calcium lactate to the polymer and/orlipid is 1:5 to 1:30.
 12. The method of claim 1, wherein thepharmaceutical composition comprises a pharmaceutically acceptableenteric coating.
 13. The method of claim 12, wherein the enteric coatingcomprises hydroxypropyl methyl cellulose phthalate (HPMCP), celluloseacetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac,polymer of methacrylic acid and an ester thereof, or combinationsthereof.
 14. The method of claim 12, wherein the weight ratio of thecalcium lactate to the enteric coating is 10:0.5 to 1:1.5.
 15. A methodof treating inflammatory disease comprising administering to a subjectin need thereof a pharmaceutical composition comprising atherapeutically effective amount of calcium lactate, wherein theinflammatory disease is selected from the group consisting ofAlzheimer's disease, and Parkinson's disease.
 16. The method of claim15, wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier, excipient, or diluent.
 17. Themethod of claim 15, wherein the pharmaceutical composition is a liquid,powder, aerosol, injection, fluid transfusion, patch, capsule, pill, ortablet, depot, or suppository.
 18. The method of claim 15, wherein thepharmaceutical composition further comprises a pharmaceuticallyacceptable polysaccharide, polymer, lipid, or combinations thereof. 19.The method of claim 18, wherein a weight ratio of the calcium lactate tothe polysaccharide is 1:<0.2 to 1:5.
 20. The method of claim 18, whereina weight ratio of the calcium lactate to the polysaccharide is 1:<0.2.21. The method of claim 15, wherein the pharmaceutical compositioncomprises a pharmaceutically acceptable enteric coating.